Somatic Genome Editing Research Articles

In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related "modifier' genes (i.e. genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.

Somatic Genome Editing: Technical Challenges and Ethical Appraisal

The ability to modify the DNA sequences with molecular tools that employ nucleases has made it possible to edit genomes. Somatic genome editing is the procedure to alter the genome of somatic cells, making the changes introduced into the nucleotide sequence not inheritable. Powerful tools have been developed for therapeutic purposes to correct monogenic inherited disorders that often cause premature death and for which effective treatment options are not available. To ensure positive impact and minimise potential harms of these techniques, require to understand their limits in addition to apply values and principles that guide their use. This study reviews technical challenges of genome editing and appraises the Ethics of its application.

Abstract 2929: Multiplexed somatic genome editing to model and investigate advanced colorectal cancer

Abstract Colorectal cancer (CRC) is among the deadliest cancers worldwide, with advanced CRC being the leading cause of death. For the development of novel therapy options, it is crucial to investigate mechanisms of advanced CRC in a bona fide way. However, little is known about the impact of distinct genomic alterations on cancer progression. The assessment of the function and role of the putative drivers in tumor progression via sequencing data remains challenging, due to the complexity and possible combinations of the candidates. Hence, genetically engineered mouse models (GEMMs) represent the mainstay for the investigation of the role of oncogenes and tumor suppressor genes in cancer progression. However, particularly in CRC, they are limited by imprecise modeling of cancer-associated changes, challenging scalability, and development of tumors in anatomically incorrect places. To tackle these issues, we developed SOmatic CRISPR-mediated genome Alterations of Tumor suppressors with Exploratory single cell Sequencing (SOCRATES) to analyze advanced CRC. We multiplexed CRISPR-Cas systems, packaged them into lentiviral particles, and delivered them to enterocytes via colonoscopy-guided submucosal injection. With this innovative approach, SOCRATES enables rapid and direct in vivo genome engineering for the modeling of advanced CRC with distinct mutational patterns and the investigation of the cancer cell states and tumor microenvironment (TME) upon editing the genes of interest. Similar to the Greek philosopher Socrates, who established a form of argumentized dialogue between individuals, SOCRATES facilitates the understanding of previously undiscovered communication between cancer cells and TME. We analyzed the influence of the most recurrent tumor suppressor mutations (Fbxw7, Ptprt, Arid1a, Atrx, Atm, Smad4, Pten) on the cancer cell state and TME in advanced CRC. Further, we created a TS-TME interaction atlas and revealed novel mechanistic insights into how the TS mutations rewire cell states and cell-cell communications. With this generated knowledge, SOCRATES will contribute to our understanding of complex tumor ecosystems and foster the improvement of diagnostic and therapeutic approaches. Citation Format: Manuel Mastel, Rene Jackstadt, Jasmin Meier, Gabriele Diamante. Multiplexed somatic genome editing to model and investigate advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2929.

Long-term efficacy and safety of cardiac genome editing for catecholaminergic polymorphic ventricular tachycardia.

Heterozygous autosomal-dominant single nucleotide variants in RYR2 account for 60% of cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia disorder associated with high mortality rates. CRISPR/Cas9-mediated genome editing is a promising therapeutic approach that can permanently cure the disease by removing the mutant RYR2 allele. However, the safety and long-term efficacy of this strategy have not been established in a relevant disease model. The purpose of this study was to assess whether adeno-associated virus type-9 (AAV9)-mediated somatic genome editing could prevent ventricular arrhythmias by removal of the mutant allele in mice that are heterozygous for Ryr2 variant p.Arg176Gln (R176Q/+). Guide RNA and SaCas9 were delivered using AAV9 vectors injected subcutaneously in 10-day-old mice. At 6 weeks after injection, R176Q/+ mice had a 100% reduction in ventricular arrhythmias compared to controls. When aged to 12 months, injected R176Q/+ mice maintained a 100% reduction in arrhythmia induction. Deep RNA sequencing revealed the formation of insertions/deletions at the target site with minimal off-target editing on the wild-type allele. Consequently, CRISPR/SaCas9 editing resulted in a 45% reduction of total Ryr2 mRNA and a 38% reduction in RyR2 protein. Genome editing was well tolerated based on serial echocardiography, revealing unaltered cardiac function and structure up to 12 months after AAV9 injection. Taken together, AAV9-mediated CRISPR/Cas9 genome editing could efficiently disrupt the mutant Ryr2 allele, preventing lethal arrhythmias while preserving normal cardiac function in the R176Q/+ mouse model of CPVT.

Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups.

Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. However, this approach requires a long and laborious task. Recently, many researchers have attempted to overcome these limitations by generating somatic mutations in the adult stage through tail vein injection or local administration of CRISPR reagents, as a new strategy called "in vivo somatic cell genome editing". This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. The newborn is an ideal stage to perform in vivo somatic cell genome editing because it is immune-privileged, easily accessible, and only a small amount of CRISPR reagents is required to achieve somatic cell genome editing throughout the entire body, owing to its small size. In this review, we summarize in vivo genome engineering strategies that have been successfully demonstrated in newborns. We also report successful in vivo genome editing through the neonatal introduction of genome editing reagents into various sites in newborns (as exemplified by intravenous injection via the facial vein), which will be helpful for creating models for genetic diseases or treating many genetic diseases.

Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Human genome editing in clinical applications: Japanese lay and expert attitudes.

Background: The world's first gene-edited babies, reported by the Chinese scientist He Jiankui, prompted an outcry of criticism and concerns worldwide over the use of genome editing for reproductive purposes. Many countries and academic associations opposed to heritable genome editing (HGE) called for public discussion involving various stakeholders. To hold a discussion of this nature and form a consensus concerning HGE, we must understand under what conditions stakeholders consider HGE acceptable and the reasons for which they deem it unacceptable. Methods: Laypeople and researchers were surveyed in May 2019. They were asked about the degree of their acceptance toward somatic genome editing (SGE) and HGE; those who answered "acceptable depending on the purpose" were queried further regarding their acceptance in the contexts of specific clinical purposes. Results: Responses were obtained from 4,424 laypeople and 98 researchers. The percentage of respondents choosing each option in attitudes to HGE was, from largest to smallest: "acceptable depending on purpose" (laypeople 49.3%; researchers 56.1%), "not acceptable for any purpose" (laypeople 45.8%; researchers 40.8%), and "acceptable for any purpose" (laypeople 5.0%; researchers 3.1%). In an additional question for those who answered "acceptable depending on the purpose," laypeople found the following purposes acceptable: infertility treatment (54.5%), treatment of life-threatening diseases (52.2%), and treatment of debilitating diseases (51.4%). Meanwhile, the degree of acceptance for enhancement purposes was 10.7, 7.9, 6.2, and 5.5% for physical, cognitive, health, and personality enhancements, respectively. In contrast, acceptance among the researchers was 94.5% and 92.7% for the treatment of life-threatening and debilitating diseases, respectively, compared with 69.1% for infertility treatment. Researchers' acceptance for enhancement purposes was similar to that of the lay participants, with 12.7, 9.1, 10.9, and 5.5% for physical, cognitive, health, and personality enhancement, respectively. Conclusion: In the past, debates regarding the acceptability of human genome editing in clinical applications tend to focus on HGE in many countries. Society will now need to debate the acceptability of both types of human genome editing, HGE and SGE.

Between desire and fear: a qualitative interview study exploring the perspectives of carriers of a genetic condition on human genome editing

Human genome editing technologies are advancing at a rapid pace, and their potential disruptive implications lead to ethical and societal questions that cannot be addressed by scientists alone. Further consideration of different stakeholders’ views on human genome editing is crucial to translate society’s needs and values into thoughtful regulations and policies. We therefore explored the views of carriers of autosomal dominant disorders on somatic and heritable genome editing (SGE and HGE) and the role of their (secular or religious) worldviews. This group of stakeholders would be most impacted by the eventual clinical application of genome editing technologies and therefore their views must be taken into account. Ten in-depth semi-structured interviews were conducted, and data were analysed using reflexive thematic analysis. We found an overarching theme: ‘Balancing between the desire to prevent serious diseases in individuals through HGE, and the fear of the harmful impact on society and nature’ and three main themes: ‘The benefits of SGE and HGE for individuals’, ‘the societal consequences of using HGE’, and ‘the consequences of interfering with nature through HGE’. Although the lived experiences of the participants varied, they were positive towards the safe use of SGE regardless of the severity of conditions, and most participants were positive towards the use of HGE but only to prevent severe genetic conditions. A few participants were against using HGE in any case, regardless of the severity of a condition, based on their religious beliefs. However, most participants with either religious or secular worldviews reported similar views on HGE, both regarding their desire to prevent serious genetic disorders and their fear of the impact on society and nature if HGE were implemented more widely. Reflecting on HGE involved complex and often ambivalent views. When engaging different stakeholders, space is needed for ambivalence and the weighing of values.

Safety and Ethical Considerations of CRISPR/Cas9-based Human Germline Genome Editing

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 adapted from natural immune defense systems of bacteria and archaea can edit genes in numerous organisms including humans. This has revolutionized genetic engineering and can help better understand genetic diseases as well as potentially correct causative mutations. Current CRISPR/Cas9-based genome editing is focused on either somatic cells or germline cells. In contrast to somatic cell genome editing, germline genome editing raises significant ethical issues as the genomic modifications in those reproductive cells by CRISPR/Cas9 can potentially be passed on to future generations. In this article, we focus on discussion of safety and ethical issues of CRISPR/Cas9-based germline genome editing in humans from several aspects including off-targets, Immunogenicity, autonomy, religion and eugenics.

Genome Editing and Cardiac Arrhythmias.

This article reviews progress in the field of cardiac genome editing, in particular, its potential utility in treating cardiac arrhythmias. First, we discuss genome editing methods by which DNA can be disrupted, inserted, deleted, or corrected in cardiomyocytes. Second, we provide an overview of in vivo genome editing in preclinical models of heritable and acquired arrhythmias. Third, we discuss recent advancements in cardiac gene transfer, including delivery methods, gene expression optimization, and potential adverse effects associated with therapeutic somatic genome editing. While genome editing for cardiac arrhythmias is still in its infancy, this approach holds great promise, especially for inherited arrhythmia syndromes with a defined genetic defect.

Abstract 2789: Tumor suppressor genotype dramatically impacts lung cancer response to KRAS G12C inhibitors in vivo

Abstract Cancer is characterized by diverse genomic alterations that change cell state and can modify therapy responses. In some cases, the relationships between genotype and drug responses are obvious, such as the response of tumors with defined oncogenic mutations to inhibitors of those mutant proteins. However, covalent KRAS G12C inhibitors (G12Ci) induce clinical responses in less than half of patients with oncogenic KRAS G12C-driven lung cancer, illustrating that genomic alterations not obviously related to the drug mechanism can have a dramatic impact. We have developed and optimized an in vivo platform to identify pharmacogenomic interactions that dictate lung cancer responses to therapies. By coupling somatic genome editing, tumor barcoding, ultra-deep barcode sequencing, and robust statistical methods in genetically engineered mouse models of human lung cancer, we can quantify and compare candidate therapies across thousands of clonal tumors of diverse tumor suppressor genotypes. This platform provides a uniquely high-throughput and quantitative assessment of tumors that are initiated de novo within the natural immunocompetent tissue environment. To uncover genotypes that are particularly important in controlling the response of tumors to oncogenic KRAS inhibition, we applied this platform to quantify the impact of 59 tumor suppressor genes on KRASG12C-driven lung tumor responses to G12Ci. Treatment resulted in approximately three-quarters reduction in both average tumor sizes and overall tumor burden, with a clear drug and dose dependence. Approximately one-quarter of inactivated genes significantly and consistently altered tumor responses across numerous G12Ci and replicate studies. Interestingly, most of these genes are thought to be in pathways not directly related to RAS signaling, and they exhibit a striking pattern in which treatment sensitivity is positively correlated with strength of the tumor suppressor effect. This pharmacogenomic profile for G12Ci was categorically different than the profiles we have observed for chemotherapy and other therapies. Overall, these results provide direct causal evidence that certain tumor suppressor genotypes dramatically shift the effectiveness of G12Ci in vivo and generate hypotheses about patients likely to benefit from G12Ci relative to alternatives. Analysis of available human data provides early clinical support for some of these hypotheses. Ongoing efforts to define pharmacogenomic profiles of combination therapies could be of even greater importance. Platforms that can accurately predict how tumor genotype drives responses have the potential to transform precision cancer therapy, enabling more effective patient stratification and therapy combinations. Citation Format: Paul J. McMurdie, Ian P. Winters, Lily M. Blair, Lafia Sebastian, Vy Tran, Gabriel Grenot, Edwin A. Apilado, Edwin A. Apilado, Ian K. Lai, Gregory D. Wall, Dmitri A. Petrov, Monte M. Winslow, Michael J. Rosen, Joseph Juan. Tumor suppressor genotype dramatically impacts lung cancer response to KRAS G12C inhibitors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2789.

Somatic Genome Editing Governance Approaches and Regulatory Capacity in Different Countries

Somatic Genome Editing Governance Approaches and Regulatory Capacity in Different Countries

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

BackgroundCamellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.ResultsThe in vitro grown seedlings of C. oleifera ‘Huashuo’ were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at − 0.07 MPa for 20 min. The maximum yield (3.5 × 107/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min.ConclusionIn summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.

Genome Editing and Pathological Cardiac Hypertrophy.

Three major genome editing tools, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeat (CRISPR) systems, are increasingly important technologies used in the study and treatment of hereditary myocardial diseases. Germ cell genome editing and modification can permanently eliminate monogenic cardiovascular disease from the offspring of affected families and the next generation, although ethically controversial. Somatic genome editing may be a promising method for the treatment of hereditary cardiomyopathy various diseases for which gene knockout is favorable and can also treat people who are already ill, although there are currently some technical challenges. This chapter describes the application of genome editing in the experimental studies and treatment of hypertrophic cardiomyopathy as well as other cardiomyopathies.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Using mouse liver cancer models based on somatic genome editing to predict immune checkpoint inhibitor responses

Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs. A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing. Based on the CD8+ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53KO/MYCOE) and a hot tumor (Keap1KO/MYCOE) were further characterized. Tumor-infiltrating CD8+ T cells from Keap1KO/MYCOE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8+ T-cell phenotype compared to those in Trp53KO/MYCOE HCCs. The TKI sorafenib sensitized Trp53KO/MYCOE HCCs to anti-PD-1 treatment. Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors. Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8+ T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8+ T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.

LPA disruption with AAV-CRISPR potently lowers plasma apo(a) in transgenic mouse model: A proof-of-concept study

Lipoprotein(a) (Lp(a)) represents a unique subclass of circulating lipoprotein particles and consists of an apolipoprotein(a) (apo(a)) molecule covalently bound to apolipoprotein B-100. The metabolism of Lp(a) particles is distinct from that of low-density lipoprotein (LDL) cholesterol, and currently approved lipid-lowering drugs do not provide substantial reductions in Lp(a), a causal risk factor for cardiovascular disease. Somatic genome editing has the potential to be a one-time therapy for individuals with extremely high Lp(a). We generated an LPA transgenic mouse model expressing apo(a) of physiologically relevant size. Adeno-associated virus (AAV) vector delivery of CRISPR-Cas9 was used to disrupt the LPA transgene in the liver. AAV-CRISPR nearly completely eliminated apo(a) from the circulation within a week. We performed genome-wide off-target assays to determine the specificity of CRISPR-Cas9 editing within the context of the human genome. Interestingly, we identified intrachromosomal rearrangements within the LPA cDNA in the transgenic mice as well as in the LPA gene in HEK293T cells, due to the repetitive sequences within LPA itself and neighboring pseudogenes. This proof-of-concept study establishes the feasibility of using CRISPR-Cas9 to disrupt LPA invivo, and highlights the importance of examining the diverse consequences of CRISPR cutting within repetitive loci and in the genome globally.

Insights into the editing of the human genome: where can novel non-viral polymeric delivery agents take us

Tom Foti has more than 25 years of biotechnology experience and serves as Vice President/GM of the Protein Business Unit. He was one of the original founders of Aldevron’s protein services business, which was formed in 2009. Prior to working for Aldevron, Foti served in several roles in the Merck KGaA Bioscience Division, most recently as the Director of its Global Custom Services Business. He started his career in 1992, with Novagen, Inc. serving in manufacturing and operational roles until 1999. He played college basketball while earning a Bachelor’s of Science in Biotechnology and Microbiology from North Dakota State University. Foti also holds a Master’s in Business Administration from Edgewood College and a Management Leadership Certificate from Massachusetts Institute of Technology.Kris Saha is an Associate Professor of Biomedical Engineering and Medical History and Bioethics at the University of Wisconsin-Madison. He was recently named the McPherson Eye Research Institute’s Retina Research Foundation Kathryn and Latimer Murfee Chair for 2019-2022. His lab is at the Wisconsin Institute for Discovery (WID), and he participates on campus in the executive committees of the Stem Cell and Regenerative Medicine Center, Robert F. Holtz Center on Science and Technology Studies, and Forward Bio Institute. Prior to his arrival in Madison, Dr. Saha studied chemical engineering and biotechnology at Cornell University, University of Cambridge, and the University of California, Berkeley. In 2007 he became a Society in Science: Branco-Weiss fellow in the laboratory of Professor Rudolf Jaenisch at the Whitehead Institute for Biomedical Research at MIT and in the Science and Technology Studies program at Harvard University with Professor Sheila Jasanoff in Cambridge, Massachusetts. At UW-Madison, major thrusts of his lab involve gene editing and cell engineering of human cells found in the retina, central nervous system, liver, and blood. He has published more than 75 scientific manuscripts, filed several patents, and received awards that include the National Science Foundation CAREER Award, Biomedical Engineering Society’s Rising Star Award, and Gund Harrington Scholar Award. He is the leader of the gene therapy biomanufacturing impact area of the Grainger Institute for Engineering, a member of the National Academies’ Forum on Regenerative Medicine, a co-lead for the T cell testbed within the National Science Foundation’s Center for Cell Manufacturing Technologies (CMaT) and on the Executive Committee of the National Institutes for Health’s Somatic Cell Genome Editing (SCGE) Consortium.

Precise somatic genome editing for treatment of inborn errors of immunity.

Rapid advances in high throughput sequencing have substantially expedited the identification and diagnosis of inborn errors of immunity (IEI). Correction of faulty genes in the hematopoietic stem cells can potentially provide cures for the majority of these monogenic immune disorders. Given the clinical efficacies of vector-based gene therapies already established for certain groups of IEI, the recently emerged genome editing technologies promise to bring safer and more versatile treatment options. Here, we review the latest development in genome editing technologies, focusing on the state-of-the-art tools with improved precision and safety profiles. We subsequently summarize the recent preclinical applications of genome editing tools in IEI models, and discuss the major challenges and future perspectives of such treatment modalities. Continued explorations of precise genome editing for IEI treatment shall move us closer toward curing these unfortunate rare diseases.

Risk assessment in gene therapy and somatic genome-editing: An expert interview study

BackgroundInnovation in gene therapy and genome editing raises high expectations for therapeutic breakthroughs. With the increasing maturity of the field, gene products using “gene transfer” technology, such as viral vectors, designer nucleases, incl. CRISPR/Cas, as the most recent, are frequently tested in clinical trials. Before such trials are launched, the anticipated risks and benefits of using gene transfer technologies must be evaluated to ascertain an ethical balance of risks and benefits. MethodsWe conducted semi-structured interviews with experts (n=15) in gene therapy/genome editing. We applied thematic text analysis to identify the qualitative spectrum of strengths, weaknesses, opportunities, and threats (SWOT) of a risk assessment approach to gene therapy/genome editing research based on a comprehensive set of nine mechanistic categories of adverse reactions combined with estimates of risk probability according to World Health Organization (WHO) adverse reaction terminology. ResultsOur study revealed a clear demand for a structured approach to risk assessment gene therapy/genome editing. The interviews indicate that the nine presented mechanistic categories may be helpful to structure this risk assessment prior to initiating a new study. The interviews revealed a broad spectrum of practice-oriented SWOT, described in detail in this manuscript. DiscussionThe here presented SWOT for a structured approach to risk assessment prior to clinical trials with gene therapy/genome editing inform the refinement and implementation of such standardized approaches and the discussion among researchers, regulators, and funders. To overcome potential weaknesses and threats in the application of such a risk-based approach, the mechanistic categories need to be case-sensitive and complemented by information on the validity of relevant animal models, long-term risks, and information about patient characteristics.