Human Germline Research Articles

Modeling genetic epileptic encephalopathies using brain organoids

Developmental and epileptic encephalopathies (DEE) are a group of disorders associated with intractable seizures, brain development, and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain‐containing oxidoreductase (WWOX) are associated with a relatively mild autosomal recessive spinocerebellar ataxia‐12 (SCAR12) and a more severe early infantile WWOX‐related epileptic encephalopathy (WOREE). In this study, we generated an in vitro model for DEEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR‐engineered human ES cells and from patient‐derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt pathway and DNA damage response impairment. Furthermore, we provide a proof of concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies.

When is it Safe to Edit the Human Germline?

In the fall of 2018 Jiankui He shocked the international community with the following announcement: two female babies, "Lulu" and "Nana," whose germlines had been modified by the cutting edge, yet profoundly unsafe CRISPR-Cas9 technology had been born. This event galvanized policy makers and scientists to advocate for more explicit and firm regulation of human germline gene editing (GGE). Recent policy proposals attempt to integrate safety considerations and public input to identify specific types of diseases that may be safe targets for human GGE (Sarkar forthcoming; Guttinger. 2019. "Editing the Reactive Genome: Towards Postgenomic Ethics of Germline Editing." Journal of Applied Philosophy. "Editing the Reactive Genome: Towards Postgenomic Ethics of Germline Editing." Journal of Applied Philosophy. "Editing the Reactive Genome: Towards Postgenomic Ethics of Germline Editing." Journal of Applied Philosophy; Lander et al., 2019). This paper argues these policy proposals are inadequate in different ways. While Sarkar (forthcoming) intends to incorporate input from the disability community for the purpose of deciding the value of human GGE, I argue that his strategy for doing so is inadequate. I'll argue that an iterative, deliberative process is a more appropriate framework for allowing the disability community to inform policy on human GGE. Further policy proposals have been framed in terms of monogenetic or single-gene diseases (Guttinger. 2019. "Editing the Reactive Genome: Towards Postgenomic Ethics of Germline Editing." Journal of Applied Philosophy. "Editing the Reactive Genome: Towards Postgenomic Ethics of Germline Editing." Journal of Applied Philosophy. "Editing the Reactive Genome: Towards Postgenomic Ethics of Germline Editing." Journal of Applied Philosophy; Lander et al., 2019). I argue that this way of conceptualizing disease is not what matters for deciding which disorders are viable candidates for human GGE. Instead, what matters is that (1) the disease in question must have (among its set of causes) genes that have a high degree of causal control with respect to the disease and (2) alternative nucleic acid sequences variants that are likely to produce traits deemed desirable must be identified. Previous policy proposals leave (2) unspecified. What conditions must be met for satisfying condition (2) should not be left to individual scientists to decide for themselves. The present proposal offers some guidance on this issue.

Taking responsibility, making irresponsibility: Controversies in human gene editing.

In current discussions, human germline editing is often called 'irresponsible'. Looking at the international summits on human gene editing held in 2015 and in 2018 and the announcement by He Jiankui of the birth of two gene-edited babies in November 2018, this article analyses how 'irresponsible' research was the result of various (dis)qualifications and demarcations. Against a background of discussions of responsibility, an individual scientist was singled out, his experiments were scrutinized for their soundness, legality and safety and ethical and moral stances were questioned. These are features of a process that I call 'irresponsibilization'. This irresponsibilization of research is entangled with calls for further action: Irresponsible research like that of He Jiankui should be contained, the veracity of knowledge claims needs to be confirmed, and institutions and decision-makers are called to act. The controversy turned 'irresponsible' into an active category, and rendered explicit its political, institutional and practical ramifications.

From goodness to good looks: Changing images of human germline genetic modification.

When writing about deliberate changes to the human germline, bioethicists tend not to discuss the modification of specific genes and instead refer to broader concepts like making people smarter, taller, or longer‐lived. Only a limited number of these traits are mentioned regularly in the literature. Examples like health and intelligence appear frequently at all stages of the germline modification discourse, but the third most frequently mentioned trait has shifted over time. Prior to the early 1980s, publications discussed giving humans a kinder temperament significantly more often than cosmetic modifications, while more recent works reverse the frequency of these traits. Contributing factors likely include a greater focus on individual decision‐making, combined with the increasing uptake of real‐world reproductive technologies like IVF and gamete donation. This shifting imagery could have a profound influence on the way scholars develop arguments about gene editing since cosmetic modifications are generally viewed more negatively and considered less relevant to the identity of future people. In comparison with earlier images of germline modification, they also suggest a more contemporary, Western, and politically liberal social context for gene editing technology. Examining how authors move between writing about different traits can also help us to be aware of the traits that are arbitrarily omitted from the discourse and to consider our preparedness for unexpected kinds of modification.

Abstract LB030: A novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19

Abstract CD19-directed chimeric antigen receptor T (CART19) cell therapies have shown impressive clinical outcomes in CD19+ B cell malignancies. All the FDA-approved CART19, including tisagenlecleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel use anti-CD19 single-chain variable fragments (scFv) that bind to the same CD19 epitope. This epitope is located at the membrane-distal portion of CD19. We aimed to develop an anti-CD19 CAR that binds to a membrane-proximal domain. To this goal, we screened a chicken immune scFv library against the CD19 extracellular domain. We selected an scFv clone (1218) that was not competing with FMC63 for CD19 binding in competition enzyme immunoassay and real-time interaction analysis. Subsequent mutagenesis assay by replacing several residues with monkey residues showed that, as compared to FMC63, the epitope of the 1218 scFv is localized in a membrane-proximal location at three-dimensional modeling (Teplyakov A., Proteins, 2018). The chicken 1218 scFv was humanized by CDR-grafting to human germline genes and backmutations (h1218 scFv) and was confirmed to be specific for CD19 in cell microarray assays and flow cytometry analysis. We, therefore, designed a CAR construct using the h1218 scFv, 41-BB costimulatory domain in a lentiviral backbone. CAR-T cells were successfully generated using the Myltenyi Prodigy platform. In vitro cytotoxicity and interferon release assays showed potent effector functions of h1218 CAR-T cells that compared positively with FMC63-based CART19. Our group recently described the occurrence of a B-cell leukemia relapse characterized by the expression of the CAR19 itself on the cell surface due to accidental transduction during manufacturing (Ruella M., Nat Med, 2018). Interestingly, h1218 but not FMC63 CART19 were able to recognize and kill FMC63 CAR19-expressing tumor cells. In vivo, h1218 CART19 cells led to complete tumor regressions in both Raji (CD19+ B-cell lymphoma) and Nalm-6 (B-cell leukemia) xenograft models. Currently, h1218 CART19 is under advanced preclinical development and will be tested in a phase I-II clinical trial for relapsed and refractory B-cell lymphoma patients. Citation Format: Ki-Hyun Kim, Ruchi P. Patel, Yong Gu Lee, Soohwan Kim, Ji-Hyeon Choi, Sung-Min Kim, Gae-Baik Kim, Jong-Ho Lee, Hyun-Jong Lee, Ji-Ho Park, Guewha Lee, LeiGuang Cui, Min Yoon, Ki Hyun Kim, Soohyun Kim, In-Sik Hwang, Youngha Lee, Bong-Kook Ko, Jong-Seo Lee, Junho Chung, Marco Ruella. A novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB030.

Thinking the unthinkable: how did human germline genome editing become ethically acceptable?

Two major reports in the UK and USA have recently sanctioned as ethically acceptable genome editing of future generations for the treatment of serious rare inherited conditions. This marks an important turning point in the application of recombinant DNA techniques to humans. The central question this paper addresses is how did it became possible for human genetic engineering (HGE) of future generations to move from an illegitimate idea associated with eugenics in the 1980s to a concrete proposal sanctioned by scientists and bioethicists in 2020? The paper uses the concept of a regime of normativity to understand the co-evolution and mutual shaping of technology, imaginaries, norms and governance processes in debates about HGE in the USA and UK. It will be argued that interlinked discursive, institutional, political and technological changes have made proposals for the use of genome editing in the genetic engineering of future generations both “thinkable” and legitimate.

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Clinical trials of germline gene editing: The exploitation problem.

The birth of the world's first genetically edited babies in 2018 provoked considerable ethical outrage. Nonetheless, many scientists and bioethicists now advocate the pursuit of clinical uses of human germline gene editing. Progress towards this goal will require research, including clinical trials where genetically edited embryos are implanted into a woman's uterus, gestated, and brought to term. This paper argues that such trials would likely conflict with the fundamental research ethical requirement of non-exploitation. This is because they would expose people who are in a vulnerable situation to risks and burdens that are substantial and not obviously offset by compensating benefits. I consider how the potential for exploitation in such trials might be mitigated, arguing that a feasible and justifiable approach is not easily found. If this analysis is correct, there is a significant ethical obstacle on the path towards clinical use of human germline gene editing.

GLEANER: a web server for GermLine cycle Expression ANalysis and Epigenetic Roadmap visualization

BackgroundGermline cells are important carriers of genetic and epigenetic information transmitted across generations in mammals. During the mammalian germline cell development cycle (i.e., the germline cycle), cell potency changes cyclically, accompanied by dynamic transcriptional changes and epigenetic reprogramming. Recently, to understand these dynamic and regulatory mechanisms, multiomic analyses, including transcriptomic and epigenomic analyses of DNA methylation, chromatin accessibility and histone modifications of germline cells, have been performed for different stages in human and mouse germline cycles. However, the long time span of the germline cycle and material scarcity of germline cells have largely limited the understanding of these dynamic characteristic changes. A tool that integrates the existing multiomics data and visualizes the overall continuous dynamic trends in the germline cycle can partially overcome such limitations.ResultsHere, we present GLEANER, a web server for GermLine cycle Expression ANalysis and Epigenetics Roadmap visualization. GLEANER provides a comprehensive collection of the transcriptome, DNA methylome, chromatin accessibility, and H3K4me3, H3K27me3, and H3K9me3 histone modification characteristics in human and mouse germline cycles. For each input gene, GLEANER shows the integrative analysis results of its transcriptional and epigenetic features, the genes with correlated transcriptional changes, and the overall continuous dynamic trends in the germline cycle. We further used two case studies to demonstrate the detailed functionality of GLEANER and highlighted that it can provide valuable clues to the epigenetic regulation mechanisms in the genetic and epigenetic information transmitted during the germline cycle.ConclusionsTo the best of our knowledge, GLEANER is the first web server dedicated to the analysis and visualization of multiomics data related to the mammalian germline cycle. GLEANER is freely available at http://compbio-zhanglab.org/GLEANER.

Leftovers of viruses in human physiology.

Significant advances have been observed in the field of cell biology, with numerous studies exploring the molecular genetic pathways that have contributed to species evolution and disease development. The current study adds to the existing body of research evidence by reviewing information related to the role of leftover viruses and/or viral remnants in human physiology. To explore leftover viruses, their incorporation, and their roles in human physiology. The study entailed conducting a systematic search in the PsycINFO, PubMed, Web of Science, and CINAHL databases to locate articles related to the topic of investigation. The search terms included “leftovers,” “viruses,” “genome sequences,” “transposable elements,” “immune response,” and “evolution.” Additional articles were selected from the references of the studies identified in the electronic databases. Evidence showed that both retroviruses and nonretroviruses can be integrated into the human germline via various mechanisms. The role of leftover viruses in human physiology has been explored by studying the activation of human retroviral genes in the human placenta, RNA transfer between neurons through virus-like particles, and RNA transfer through extracellular vesicles. Research evidence suggested that leftover viruses play key roles in human physiology. A more complete understanding of the underlying pathways may provide an avenue for studying human evolution and allow researchers to determine the pathogenesis of some viral infections. Evidence obtained in this review shows that leftover viruses may be incorporated into the human genome. Retroviral genes are critical for the development of different parts of the body, such as the placenta in mammals.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00429-021-02306-8.

He Jiankui´s gene-editing experiment and the non-identity problem.

Genetic engineering has been a topic of discussion for over 50 years, but it is only recently that gene editing has become a reality. CRISPR biotechnologies have made gene editing much safer, precise and feasible. We have witnessed the first cases of human germline genetic modification resulting in live births, conducted by He Jiankui. In this paper, we will analyse He Jiankui’s case in relation to one of the most difficult problems in procreative ethics (or the ethics of future generations): the non‐identity problem. We believe that this analysis will help us to understand the ethics involved in gene editing and hopefully allow for a better, more philosophically grounded legislation on CRISPR and other gene‐editing technologies.

Gene Editing: How Can You Ask "Whether" If You Don't Know "How"?

Though questions about whether gene editing should be done at all have dominated ethical discussion, a literature about how it can be done ethically has been growing. Work on responsible translational pathways for human germline gene editing has been criticized for focusing on the wrong questions. But questions about responsible translational pathways-questions about how gene editing could be done ethically-are, in an important sense, prior to questions about whether it is desirable and permissible. Asking "whether" questions about gene editing requires a model of what responsible clinical use of gene editing would look like.

PL03.3: Ethical considerations related to human germline editing

PL03.3: Ethical considerations related to human germline editing

Neuronal deletion of Wwox, associated with WOREE syndrome, causes epilepsy and myelin defects.

WWOX-related epileptic encephalopathy (WOREE) syndrome caused by human germline bi-allelic mutations in WWOX is a neurodevelopmental disorder characterized by intractable epilepsy, severe developmental delay, ataxia and premature death at the age of 2-4 years. The underlying mechanisms of WWOX actions are poorly understood. In the current study, we show that specific neuronal deletion of murine Wwox produces phenotypes typical of the Wwox-null mutation leading to brain hyperexcitability, intractable epilepsy, ataxia and postnatal lethality. A significant decrease in transcript levels of genes involved in myelination was observed in mouse cortex and hippocampus. Wwox-mutant mice exhibited reduced maturation of oligodendrocytes, reduced myelinated axons and impaired axonal conductivity. Brain hyperexcitability and hypomyelination were also revealed in human brain organoids with a WWOX deletion. These findings provide cellular and molecular evidence for myelination defects and hyperexcitability in the WOREE syndrome linked to neuronal function of WWOX.

What can evolutionary biology learn from cancer biology?

Detecting and treating cancer effectively involves understanding the disease as one of somatic cell and tumor macroevolution. That understanding is key to avoid triggering an adverse reaction to therapy that generates an untreatable and deadly tumor population. Macroevolution differs from microevolution by karyotype changes rather than isolated localized mutations being the major source of hereditary variation. Cancer cells display major multi-site chromosome rearrangements that appear to have arisen in many different cases abruptly in the history of tumor evolution. These genome restructuring events help explain the punctuated macroevolutionary changes that mark major transitions in cancer progression. At least two different nonrandom patterns of rapid multisite genome restructuring – chromothripsis (“chromosome shattering”) and chromoplexy (“chromosome weaving”) – are clearly distinct in their distribution within the genome and in the cell biology of the stress-induced processes responsible for their occurrence. These observations tell us that eukaryotic cells have the capacity to reorganize their genomes rapidly in response to calamity. Since chromothripsis and chromoplexy have been identified in the human germline and in other eukaryotes, they provide a model for organismal macroevolution in response to the kinds of stresses that lead to mass extinctions.

The Brave New World: Should we tread down the path to human germline editing?

Gene-editing tools such as the CRISPR-Cas9 system create an opportunity for individuals to have their DNA edited for specific purposes. Somatic cell editing targets specific cells in an individual, and is aimed at providing a therapeutic mechanism to correct a genetic disease or condition. Germline editing refers to the editing of the DNA of embryos or gametes, which creates edits that are heritable. Following the announcement in 2018 that the Chinese scientist He Jiankui had proceeded to gene-edit human embryos, a moratorium on germline editing was quickly proposed. While an objective of the moratorium was to prevent clinical application of germline editing, it also served as an opportunity to engage in global debate on the issues inherent in gene editing. Heritable editing has become an ethically controversial topic in bioethics, and this article undertakes to provide a primer in the existing national and international legal framework for gene editing, as well as description of the prominent current views of heritable germline editing.

Human Germline Genome Editing: On the Nature of Our Reasons to Genome Edit

Ever since the publication of Derek Parfit’s Reasons and Persons, bioethicists have tended to distinguish between two different ways in which reproductive technologies may have implications for the welfare of future persons. Some interventions harm or benefit particular individuals: they are “person affecting.” Other interventions determine which individual, of a number of possible individuals, comes into existence: they are “identity affecting” and raise the famous “non-identity problem.” For the past several decades, bioethical debate has, for the most part, proceeded on the assumption that direct genetic modification of human embryos would be person affecting. In this paper, I argue that that genome editing is highly unlikely to be person affecting for the foreseeable future and, as a result, will neither benefit nor harm edited individuals.

Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.

Gene therapies for genetic diseases have been sought for decades, and the relatively recent development of the CRISPR/Cas9 gene-editing system has encouraged a new wave of interest in the field. There have nonetheless been significant setbacks to gene therapy, including unintended biological consequences, ethical scandals, and death. The major focus of research has been on technological problems such as delivery, potential immune responses, and both on and off-target effects in an effort to avoid negative clinical outcomes. While the field has concentrated on how we can better achieve gene therapies and gene editing techniques, there has been less focus on when and why we should use such technology. Here we combine discussion of both the technical and ethical barriers to the widespread clinical application of gene therapy and gene editing, providing a resource for gene therapy experts and novices alike. We discuss ethical problems and solutions, using cystic fibrosis and beta-thalassemia as case studies where gene therapy might be suitable, and provide examples of situations where human germline gene editing may be ethically permissible. Using such examples, we propose criteria to guide researchers and clinicians in deciding whether or not to pursue gene therapy as a treatment. Finally, we summarize how current progress in the field adheres to principles of biomedical ethics and highlight how this approach might fall short of ethical rigour using examples in the bioethics literature. Ultimately by addressing both the technical and ethical aspects of gene therapy and editing, new frameworks can be developed for the fair application of these potentially life-saving treatments.

Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes.

Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. Recently, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Using VH3–23 as a model framework, we observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Our observations shed light on the design of next-generation synthetic diversity with improved probability of success.