Although there have been advancements with mRNA delivery using LNPs, finding ionizable lipids that can work across a variety of therapeutic uses has been a roadblock. While some lipids have been effective—like those used in COVID-19 vaccines—they’re often optimized for specific applications, such as targeting the liver. To make mRNA therapies viable for treating more diseases, we need lipids that perform well in different tissues without causing unwanted immune responses. The problem is, there’s an enormous number of possible lipid structures, and manually exploring each one is both time-consuming and costly. So, there’s a real need for a way to speed up the discovery of these essential lipids.

Recent paper published in Nature Materials and performed by Assistant Professor Roy van der Meel, Francesca Grisoni & Professor Willem Mulder from the Eindhoven University of Technology, tackles a crucial aspect of mRNA-based therapies: figuring out how to get mRNA safely and effectively into cells. mRNA therapies have shown a lot of promise, especially for vaccines and gene treatments, but they rely on delivering fragile mRNA molecules without breaking them down along the way. That’s where lipid nanoparticles (LNPs) come in. These tiny carriers protect mRNA as it navigates through the body’s barriers to reach target cells. A vital part of these nanoparticles is the ionizable cationic lipids, which bind to mRNA and help it get into cells. But, finding the right lipids that work well and are safe is a tough challenge.

To tackle this, the researchers turned to machine learning and high-throughput synthesis. By harnessing the predictive power of computational models, they aimed to save time and resources when identifying promising lipid candidates. Their approach combined machine learning with a method called the Ugi four-component reaction, which allowed them to quickly create a large library of lipids. This method enabled them to predict which lipids might offer high transfection efficiency and a low immune reaction before even stepping into the lab for extensive testing. The motivation here was straightforward: by streamlining the discovery of new ionizable lipids, the researchers hoped to unlock mRNA’s full potential, making it more accessible and adaptable for treating various diseases. Through this innovative strategy, they not only aimed to enhance delivery methods but also to expand mRNA’s therapeutic reach. This could potentially open up mRNA therapies for targeting more than just the liver, extending to the lungs, muscles, and other tissues as well.

The researchers carried out a series of experiments to find better ionizable lipids for mRNA delivery, focusing on both the discovery and effectiveness of these lipids. To start, they used a chemical reaction process known as the Ugi four-component reaction. This technique allowed them to quickly generate a library of 384 unique ionizable cationic lipids. Once they had these lipids, they formulated them into lipid nanoparticles (LNPs) containing luciferase mRNA—a marker that makes it easy to track transfection efficiency in cells. Testing these formulations in vitro, they assessed how well each lipid could deliver mRNA by observing bioluminescence, a glow that indicates successful delivery. The experiments revealed several lipids with high transfection efficiency, showing the researchers which candidates might be worth exploring further. Building on these initial findings, the team wanted to broaden the chemical diversity of their lipid library. They synthesized an additional 200 lipids and then evaluated these in the same way, bringing the total number of tested lipids to 584. This expanded dataset enabled them to train a machine learning model to predict which lipid structures might work best for mRNA delivery. By using the transfection results from all these trials, they developed a model that could estimate how effective untested lipids might be based solely on their chemical makeup. The model’s predictions then guided them to screen a vast virtual library of 40,000 lipids. They synthesized 16 promising lipids based on these predictions, which was a much smaller and more manageable number to test in actual experiments.

Among the newly synthesized lipids, one candidate—referred to as 119-23—consistently showed impressive results. When the team tested LNPs formulated with 119-23 in mice, they compared its performance to well-established lipids like MC3 and SM102, which are commonly used in clinical mRNA and siRNA delivery. The experiments revealed that LNPs containing 119-23 led to higher gene expression levels after intramuscular injection than the formulations based on MC3 or SM102. Specifically, they found that 119-23 did a better job of delivering mRNA encoding for human erythropoietin (hEPO), suggesting its potential as a powerful tool for therapeutic applications.

Further experiments evaluated how well 119-23 could target different tissues. The researchers delivered LNPs containing 119-23 to the lungs via intravenous injection. In these trials, 119-23 outperformed the standard lipids once again, showing higher transfection efficiency in lung tissue. The findings suggested that 119-23 was not only effective for localized delivery but could also be suitable for systemic treatments, making it a versatile candidate for mRNA-based therapies. The team further tested its capability for organ targeting using reporter mice models. These tests confirmed that 119-23 could transfect a range of cell types within tissues like the liver, spleen, and lungs, underscoring its potential adaptability for diverse therapeutic needs.

Overall, the researchers’ experiments demonstrated that 119-23 could provide a more effective and versatile solution for mRNA delivery compared to conventional lipids. The use of machine learning, coupled with high-throughput synthesis, streamlined the process of finding such an impactful lipid, underscoring the value of this approach for future nanomedicine development. The study highlighted how computational tools and experimental work could come together to accelerate the discovery of crucial materials in medical research.  The significance of this study lies in its potential to revolutionize how we approach mRNA-based therapies. By leveraging machine learning and high-throughput synthesis, the researchers not only sped up the discovery process for ionizable lipids but also broadened the range of therapeutic applications for mRNA delivery. This is especially important given that mRNA technology has vast potential beyond the liver-focused applications we commonly see, such as in COVID-19 vaccines. With the discovery of lipid 119-23, which demonstrated superior delivery capabilities across multiple tissues, the study suggests that it’s possible to develop more adaptable and effective mRNA therapies for a broader spectrum of diseases. Another major implication is the way this approach could impact drug development. Traditionally, identifying viable lipid candidates for mRNA delivery involved lengthy, resource-intensive trials. The integration of machine learning models into this process means that researchers can now predict the effectiveness of new lipids much more efficiently, saving both time and resources. This not only accelerates the discovery process but also makes it more feasible to tailor lipid nanoparticles for specific treatments, possibly even personalizing mRNA therapies for individual patients in the future. Moreover, the success of this study highlights the growing importance of computational tools in medical research. Machine learning can handle complex datasets and detect patterns that may be overlooked in traditional experimental setups. As more high-quality data becomes available, such computational approaches could allow us to reverse-engineer LNP formulations to achieve desired effects, such as targeting specific organs or minimizing immune responses. This suggests that the field of nanomedicine is moving toward a future where we can fine-tune mRNA delivery systems with unprecedented precision, ultimately making treatments safer and more effective for patients. Finally, the broader implications of this study extend to the potential for mRNA therapies to address a wider array of diseases. The ability of lipid 119-23 to deliver mRNA to different tissues means that we may be able to target conditions beyond just genetic and infectious diseases, potentially expanding into areas like cancer, metabolic disorders, and even regenerative medicine. This versatility could transform the way we think about and develop treatments, as it opens the door for mRNA therapies to become a cornerstone of personalized medicine.

The T cell receptor (TCR) is a composite of multiple proteins with two main functions: antigen recognition (mediated by TCRα and TCRβ chains) and intracellular signaling (triggered by the CD3γ, CD3δ, CD3ε and CD3ζ chains) leading to cell activation. In contrast, a chimeric antigen receptor (CAR) is a synthetic receptor that comprises an extracellular antigen recognition domain and one or more intracellular signaling domains. CAR-based cell therapy targets cell surface tumor antigens with NK or T cells that are genetically modified to express a CAR, enabling them to target and kill cancer cells. In turn, intracellular antigens presented by the major histocompatibility complex (MHC) class I on the surface of tumor cells are recognized by TCRs. TCR adoptive cell therapy (ACT) is typically based on autologous CD8 T lymphocytes, but allogenic NK cells can be adapted to express a TCR, if they are also transfected with CD3 and CD8 genes.

Human papillomavirus type 16 (HPV16), is an oncogenic virus that causes cervical cancer and other anogenital malignancies as well as head and neck carcinomas. The HPV16 E6 and E7 genes encode oncoproteins that promote degradation of TP53 and inactivate RB, respectively, and their continuous expression is necessary for tumor progression, which makes them ideal targets for ACT.

However, the effectiveness of TCR-modified immune cells as a standalone therapy against solid tumors, like cervical cancer, has been limited. Eventually, the MHC of cancer cells does not present enough of the E6 and E7 oncoproteins to be effectively recognized and targeted by the modified immune cells. Moreover, solid tumors often create a microenvironment that is hostile to immune cells and suppress their activity, making them less effective at targeting and destroying cancer cells. While the strategy of targeting HPV16 E6 and E7 oncoproteins is appealing, the practical implementation faces significant limitations. There is a need for new approaches to improve ACT, potentially combining TCR-based cell therapy with other strategies.

One way to overcome these limitations is shown in a new study published in Journal Cancer Letters by Dr. Mansour Poorebrahim, Dr. Isaac Quiros-Fernandez (both contributing equally to the study) and Dr. Angel Cid-Arregui from the Targeted Tumor Vaccines and Applied Tumor Immunity group, German Cancer Research Center (DKFZ), Heidelberg, Germany together with Dr. Frederik Marmé from Heidelberg University and Dr. Stefan Burdach from Technical University of Munich. The researchers devised a strategy that combined a TCR specific to the HPV16 E7 oncoprotein (E7-TCR) with a CAR targeting TROP2, a cell surface antigen overexpressed in various types of cancer, including HPV-associated cancers. This was shown by a meta-analysis of the gene expression profile of cervical cancer samples, available in the Gene Expression Omnibus (GEO) public database, which was compared with the gene expression data of healthy tissues.

In their study, they constructed a gene coding a CAR against TROP2, which comprised the human CD8a leader signal peptide, a Myc tag, the scFv of an anti-TROP2 antibody, and the intracellular signaling domains of CD28 and 4-1BB. Then they used a lentiviral vector to transduce Jurkat J76 and NK-92 cell lines with the TROP2-CAR gene. In addition, they synthesized human CD3 and CD8 synthetic genes that were needed to generate NK-92 cells stably expressing all CD3 and CD8 subunits, which are required for proper surface expression and functionality of the E7-TCR in the NK-92 cells. Furthermore, primary CD8 T cells were electroporated with plasmids carrying the E7-TCR and T-CAR to test their activation capacity upon co-culture with HPV16+ and TROP2+ tumor cells. Various assays were conducted to measure cell-cell binding affinity, avidity and activation of the effector cells upon co-culture with tumor cells, and the cytotoxic capacity of the NK-92/CD3/CD8 cell line and the engineered primary CD8 T cells expressing the E7-TCR and T-CAR constructs.

The authors demonstrated that NK-92/CD3/CD8 cells expressing both E7-TCR and TROP2-CAR have significantly enhanced antigen-specific activation and cytotoxicity against HPV16+ tumor cells. The TROP2-CAR design focused on targeting a tumor-associated antigen, thereby reducing the risk of on-target off-tumor toxicity. The experiments with primary CD8 T cells isolated from healthy donors showed enhanced activation upon co-culture with HPV16+/TROP2+ tumor cells compared with controls, corroborating the efficacy of the dual TCR/CAR strategy.

The integration of TCR and CAR technologies in NK cells represents a promising advancement in cancer immunotherapy. This study showing enhancing the activation and cytotoxicity of engineered immune cells against HPV16+ tumors not only highlights the potential of this approach in treating cervical and other HPV-associated cancers but also sets a precedent for future research in the field of adoptive cell therapy. The dual receptor approach, targeting both a viral antigen and an overexpressed tumor antigen, could be a paradigm shift in the immunotherapy of solid tumors, mitigating some of the limitations of current immunotherapeutic strategies.

Argininosuccinate lyase deficiency (ASLD) is a rare inherited disorder characterized by the body’s inability to properly break down argininosuccinate due to mutations in the ASL gene. This deficiency disrupts the urea cycle, leading to the accumulation of ammonia in the bloodstream, which can cause severe neurological damage and even death if not properly managed. The traditional treatment modalities for ASLD, which include dietary restrictions, supplementation, and in severe cases, liver transplantation, offer only symptomatic relief without addressing the root cause of the disorder. Thus, the development of gene therapy approaches represents a significant advancement in the potential treatment of ASLD and similar genetic disorders. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and gene therapy represent groundbreaking approaches in the treatment of genetic disorders. These technologies offer the potential to correct the underlying genetic errors that cause such conditions, providing a more definitive solution than traditional treatments.  For instance CRISPR-Cas9 is a gene-editing tool that allows scientists to cut DNA at a specific location and either remove, add, or replace the DNA in that location. In argininosuccinate lyase deficiency, CRISPR-Cas9 system could be designed to target the specific mutations in the gene that codes for argininosuccinate lyase. By correcting these mutations, the normal function of the enzyme could be restored, allowing the urea cycle to proceed properly and prevent the accumulation of ammonia. Alternatively, a new, functional copy of the gene could be inserted into the genome of liver cells, compensating for the defective gene and restoring enzyme function.

One of the major challenges of using CRISPR and gene therapy is ensuring that the gene editing or gene insertion does not unintentionally alter other parts of the genome, which could potentially lead to unwanted side effects or new health issues. Moreover, the body’s immune system might recognize the viral vectors used in gene therapy as foreign invaders, leading to an immune response that could reduce the therapy’s effectiveness or cause other complications. Furthermore, the long-term effects and stability of gene edits or inserted genes need further study. Continuous monitoring and research are essential to ensure their safety and effectiveness over time. Indeed, CRISPR and gene therapy hold promise for treating argininosuccinate lyase deficiency and other similar genetic disorders by addressing the root cause of the disease.  To this account, a new study published in the American Journal of Human Genetics and conducted by Sami Jalil, Timo Keskinen, Juhana Juutila, Rocio Sartori Maldonado, Liliya Euro, Anu Suomalainen, Risto Lapatto, Emilia Kuuluvainen, Ville Hietakangas, Timo Otonkoski, Mervi Hyvönen, and Kirmo Wartiovaara from Helsinki University in Finland, the team developed an innovative approach aimed at addressing ASLD through a novel gene therapy approach using CRISPR technology.

They generated human-induced pluripotent stem cells (hiPSCs) from ASLD Patients where skin biopsies from two individuals homozygous for the Finnish founder ASL variant (c.1153C>T [p.Arg385Cys]) were reprogrammed to generate hiPSCs. This variant is known for its severe impact on the ASL enzyme’s function, critical in the urea cycle. This way they reported the successful generation and verification of hiPSCs provided a patient-specific model for studying ASLD in vitro, allowing for the examination of disease mechanisms and therapeutic interventions. The researchers used adenine base editors (ABEs) to specifically target and correct the c.1153C>T mutation in the hiPSCs. Edited cells were then differentiated into hepatocyte-like cells to assess functional recovery. They found a significant reduction in argininosuccinate levels was observed in edited cells compared to non-edited controls, indicating the restoration of ASL function and, by extension, the urea cycle. The team tested three different FDA-approved LNP formulations for their efficiency in delivering the ABE-encoding RNA and the sgRNA targeting the ASL variant into fibroblasts derived from ASLD patients. They found all three LNP formulations successfully delivered the CRISPR components and achieved efficient editing of the ASL variant with no apparent cell toxicity. The best performing formulations significantly reduced argininosuccinate levels to those seen in healthy donors, showcasing a potential route for clinical application.

Afterward they conducted comprehensive analyses to evaluate the potential off-target effects of the CRISPR editing and the cytotoxicity of the LNP formulations. The team observed minimal off-target effects, and the LNP formulations demonstrated a favorable safety profile, supporting the potential of this approach for safe clinical use. Moreover, the researchers analyzed the metabolic impact of correcting the ASLD mutation in hepatocyte-like cells derived from the edited hiPSCs. This included measuring the levels of argininosuccinate and other relevant metabolites. Edited hepatocyte-like cells showed a significant normalization of metabolic profiles, with a drastic reduction in argininosuccinate levels, further validating the functional correction of the urea cycle.

The authors effectively used human-induced pluripotent stem cells (hiPSCs) derived from individuals with ASLD to model the disorder in vitro. They also employed CRISPR-Cas9 technology, specifically ABEs, to target the disease-causing genetic variant in ASL, achieving a remarkable correction of the genetic defect. The differentiation of these edited hiPSCs into hepatocyte-like cells and the subsequent demonstration of normalized argininosuccinate levels validate the efficacy of this gene editing approach. Importantly, the researchers addressed a critical aspect of therapeutic application by testing various FDA-approved lipid nanoparticle formulations for delivering the gene editing components, thereby highlighting a viable pathway for in vivo application of this therapy.

The study’s focus on minimizing off-target effects and ensuring the specificity of the gene editing process underscores the careful consideration of the potential clinical implications of this therapy. The use of CRISPR technology in this manner has the potential to offer a permanent cure for ASLD by directly addressing the genetic cause of the disease, as opposed to merely managing its symptoms. Additionally, this approach could be applicable to a wide range of genetic disorders beyond ASLD, where specific genetic mutations have been identified as the cause of the disease.

In conclusion, the research conducted by Helsinki University scientists is an important step forward in the treatment of ASLD and potentially other genetic disorders. By combining cutting-edge CRISPR technology with an innovative delivery mechanism, it paves the way for future clinical applications that could offer hope to patients with ASLD and other similar genetic conditions. The implications of this research extend beyond ASLD, offering insights into the broader field of genetic medicine and the ongoing quest to develop more effective, targeted treatments for genetic diseases.

Hypomyelinating Neuropathy-3 (CHN3) is a rare neurological disorder characterized by the onset of neurogenic muscle impairment that occurs in utero. The disorder results from homozygous or compound heterozygous mutations in the CNTNAP1 gene located on chromosome 17q21. CNTNAP1 encodes Contactin-associated protein 1 (Caspr), a crucial component of the paranodal junctions in myelinated neurons, which play a critical role in maintaining the integrity of myelinated axons and facilitate saltatory conduction. Mutations in CNTNAP1 have been implicated in a spectrum of neurological disorders, characterized by disrupted nerve conduction and motor function due to axonal and myelin anomalies. Symptoms can include delayed motor development, muscle weakness, poor muscle tone, impaired muscle coordination, absence of reflexes, and difficulty in walking or crawling. In some cases, infants may experience respiratory problems or difficulty in swallowing. The exact cause of CHN3 is related to mutations in genes involved in the process of myelin formation, but the precise mechanisms by which these mutations lead to the symptoms of the disorder are not fully understood. To this account, a new study published in Cell Reports conducted by Cheng Chang, Lacey Sell, Assistant Professor Qian Shi, and led by Professor Manzoor Bhat from the University of Texas Health Science Center San Antonio, the researchers investigated the effects of CNTNAP1 mutations on nerve conduction and motor function, and explored the potential of gene therapy in rescuing these deficits. The study focused on two key mutant mouse models, Cntnap1C324R and Cntnap1R765C, which mimic human CNTNAP1 mutations. The authors used CRISPR-Cas9 technology to introduce specific point mutations into the mouse Cntnap1 gene to create two mutant lines, Cntnap1C324R and Cntnap1R765C, corresponding to the human CNTNAP1C323R and CNTNAP1R764C mutations. This genetic manipulation aimed to replicate the human disease in mice for detailed study. They evaluated the mutant mice for any signs of neurological dysfunction and compared to wild-type controls. The team found both Cntnap1C324R and Cntnap1R765C mutants exhibited significant weight loss, reduced nerve conduction velocities, and progressive motor impairments, including difficulty in maintaining balance and coordination. These phenotypes mirrored the clinical manifestations of CNTNAP1-related neuropathies in humans, establishing the validity of the mouse models for further investigation. Moreover, the authors provided compelling evidence of the detrimental impact of CNTNAP1 mutations on the paranodal structure and function. They found that mutations lead to hypomyelination and notable ultrastructural defects at the paranodes, including everted myelin loops and disrupted axo-glial junctions. These structural alterations compromise the electrical insulation of axons and impede nerve signal propagation, elucidating the basis for the observed neurological deficits. A critical finding of the research by Professor Manzoor Bhat and team is the altered stability and localization of the mutant Cntnap1 proteins. Both C324R and R765C mutants exhibit reduced protein stability and are aberrantly retained within the neuronal soma, failing to reach the paranodal regions where they are functionally required. The mislocalization is likely caused by improper folding or trafficking of the mutant proteins, underscoring the importance of precise protein processing and transport in neuronal function. Perhaps the most promising aspect of the authors’ findings is the demonstration that neuronal expression of wild-type Cntnap1 can rescue the neurological phenotypes in the mutant mice. This finding confirms the loss-of-function nature of the CNTNAP1 mutations and opens up new avenues for gene therapy. It may be possible to restore normal paranodal structure, nerve conduction, and motor function, offering hope for therapeutic interventions in humans suffering from CNTNAP1-related neuropathies by reintroducing the functional gene into affected neurons. In a nutshell, the study led by Professor Manzoor Bhat and his colleagues provided critical understanding into the pathogenic mechanisms of CNTNAP1 mutations and highlighted the potential of gene therapy for treating associated neurological disorders. The research also demonstrated the importance of timely intervention and suggest that future research should focus on optimizing gene delivery methods, assessing long-term efficacy and safety, and exploring combinatory therapeutic approaches.

Sickle cell disease (SCD) is a group of inherited red blood cell disorders. It’s characterized by the production of an abnormal form of hemoglobin, called hemoglobin S (HbS). This abnormal hemoglobin causes red blood cells to become rigid, sticky, and misshapen, resembling a crescent or “sickle” shape. SCD is caused by a mutation in the HBB gene, which provides instructions for making beta-globin, a component of hemoglobin. This mutation is inherited in an autosomal recessive pattern, meaning that a child must inherit two copies of the mutated gene (one from each parent) to be affected. If only one copy is inherited, the individual is a carrier (commonly referred to as having sickle cell trait) and usually doesn’t show symptoms. SCD is often diagnosed through blood tests, which can detect the presence of HbS. Newborn screening for SCD is standard in many countries, allowing early diagnosis and intervention. There’s no cure for SCD, but treatments can alleviate symptoms and reduce complications. Research in gene therapy and other novel treatments continues to advance, offering hope for more effective treatments or a potential cure in the future. Because SCD is a monogenic disorder it has been a prime candidate for gene therapy. Early clinical trials have shown promise in using gene therapy to induce the production of fetal hemoglobin, thereby ameliorating the symptoms of SCD. However, the recent cases of myeloid malignancies in gene therapy recipients have cast a shadow over this optimism, necessitating a deeper understanding of the genomic consequences of gene therapy in SCD patients. A new study published in Nature Medicine led by Professor Peter Campbell at Wellcome Sanger Institute & Professor David Kent from York University highlights a complex interplay between gene therapy and hematopoietic stem cell (HSC) dynamics, particularly concerning the emergence of myeloid malignancies.

The research team employed whole-genome sequencing to track the clonal evolution of HSCs in six SCD patients undergoing gene therapy. This approach enabled the detailed mapping of somatic mutations and clonal dynamics in both gene-modified and unmodified HSCs, pre- and post- gene therapy. The patient cohort, part of a clinical trial (NCT03282656), received gene therapy via plerixafor-mobilized CD34+ cells transduced with a vector inducing γ-globin expression. The researchers found a variable mutation burden in pre- gene therapy HSCs, with some patients showing elevated levels compared to age-matched controls. This raised the possibility of SCD or its treatment (like hydroxycarbamide) influencing mutation rates. Interestingly, post- gene therapy, there was an increase in potential driver mutations (particularly DNMT3A and EZH2 mutations) in both modified and unmodified cells, indicating a positive selection of mutant clones during gene therapy.

The study did not identify clonal expansions in the post- gene therapy period, a reassuring finding considering the risk of leukemogenesis. However, the increased frequency of driver mutations post- gene therapy is concerning. It suggests that the gene therapy process, including cell manipulation and engraftment, might confer a selective advantage to HSCs with pre-existing mutations, potentially heightening the risk of clonal expansions and myeloid neoplasms. An important consideration is the long-term behavior of these mutant clones. While the study’s timeframe did not allow for conclusive observations, the potential for these clones to contribute to myeloid malignancies cannot be ignored. This necessitates ongoing surveillance and research into the mechanisms driving clonal expansion in the context of gene therapy.

The findings pose significant implications for patient selection and monitoring in gene therapy trials. Screening for pre-existing driver mutations might become crucial, although the sensitivity of current technologies is a limiting factor. Furthermore, this study emphasizes the need for minimizing additional mutations pre- gene therapy, possibly advocating for earlier intervention. This research underscores the necessity for a holistic understanding of gene therapy impacts, particularly in the context of clonal hematopoiesis and mutation dynamics. Future studies should focus on identifying the specific aspects of the gene therapy process that favor clonal selection and expansion, which could lead to more refined and safer gene therapy approaches.

The study by Campbell and Kent marks a significant step in understanding the genomic landscape of SCD in the context of gene therapy. While it offers reassurance in some aspects, it also raises critical questions about the long-term implications of gene therapy, particularly regarding the selection and expansion of mutant HSC clones. These findings are pivotal in guiding future gene therapy trials, emphasizing the importance of comprehensive genomic surveillance and the need for continuous refinement of gene therapy methodologies. As we stride towards a cure for SCD and other genetic disorders, it is imperative that we balance the promise of these advanced therapies with an understanding of their complex biological implications. This research not only contributes to the field of SCD treatment but also serves as a valuable reference point for the broader application of gene therapy in various hematological conditions.

Acute Myeloid Leukemia (AML) is a type of cancer that affects the blood and bone marrow. It’s characterized by an overproduction of immature white blood cells, known as myeloblasts or leukemic blasts. These abnormal cells crowd out normal blood cells, leading to a variety of symptoms and complications.  AML starts in the bone marrow, the soft inner part of certain bones where new blood cells are made. It can quickly move into the blood and sometimes spread to other parts of the body like the lymph nodes, liver, spleen, central nervous system, and testicles in males. There are several subtypes of AML, classified based on the type of cell from which the leukemia developed and its level of maturity. This classification is important for determining the prognosis and treatment plan. AML is diagnosed through blood tests, bone marrow aspiration and biopsy, cytogenetic analysis, and other laboratory tests to identify specific genetic mutations and changes. Treatment often involves chemotherapy, targeted therapy, and sometimes stem cell transplant. AML is characterized by the rapid proliferation of undifferentiated myeloid cells, often exacerbated by genetic mutations and cytogenetic aberrations. The current standard treatments for AML, including high-dose anthracycline and cytarabine, achieve high remission rates but are often associated with significant toxicity and a notable risk of relapse. As a result, there is an urgent need for novel, targeted therapies with lower inherent toxicity, particularly for elderly patients and for maintaining remission post-treatment. The specific treatment plan depends on various factors including the subtype of AML, the patient’s age, overall health, and genetic mutations present in the cancer cells. The outlook for AML patients varies based on several factors. Age, overall health, specific genetic mutations in the leukemia cells, and how well the cancer responds to treatment are crucial in determining the prognosis. Generally, younger patients with AML tend to have a better prognosis than older adults. Research continues to make advances in understanding AML, leading to the development of new treatments and therapies. For example, targeted therapies that focus on specific genetic changes in leukemia cells have become more common.

In a new study published in the Journal Leukemia by Signe Neldeborg, Johannes Frasez Soerensen, Charlotte Thornild Møller, Marie Bill, Zongliang Gao, Rasmus O. Bak, Kasper Holm, Boe Sorensen, Mette Nyegaard, Yonglun Luo, Peter Hokland, Magnus Stougaard, Maja Ludvigsen, and led by Professor Christian Kanstrup Holm from the Aarhus University in Demnark focused on AML, specifically targeting the t(8;21) fusion found in a substantial fraction of AML cases. The study showcases a groundbreaking dual intron-targeting CRISPR-Cas9 treatment strategy that can efficiently disrupt fusion genes without needing to pinpoint the precise breakpoint location. This method has demonstrated impressive efficacy in vitro, substantially reducing growth rate and proliferation in AML t(8;21) Kasumi-1 cells, and has also shown promising results in vivo.

The authors’ approach is based on the CRISPR-Cas9 gene editing system, which has shown promise in a range of genetic diseases. By targeting introns adjacent to the fusion breakpoint of the RUNX1-RUNX1T1 gene in AML t(8;21), the researchers circumvent the need for precise breakpoint identification. This strategy effectively disrupts the oncogenic driver while preserving the integrity of the wild-type RUNX1 and RUNX1T1 genes. The method is designed to be adaptable across patients, regardless of individual genetic variations in the breakpoint region. The researchers demonstrated this technique’s efficacy using the Kasumi-1 cell line model, where they observed significant reductions in cell proliferation and tumor growth in vitro. They also validated the approach in primary cells isolated from a patient with AML t(8;21), reinforcing the potential of this method in clinical settings. Additionally, the study highlights the importance of careful consideration regarding off-target effects and the feasibility of delivering CRISPR-Cas9 components in vivo. This study represents a critical step forward in the development of targeted gene therapies for AML and potentially other cancers characterized by specific genetic aberrations. The dual intron-targeting CR strategy provides a more precise and potentially safer approach to gene editing in cancer treatment. The ability to disrupt fusion genes without needing detailed knowledge of the breakpoint location is a significant advancement, allowing for broader applicability across patients with AML t(8;21). This approach could revolutionize the treatment paradigm for AML, offering a more targeted and less toxic alternative to traditional chemotherapy.

The implications of this study extend beyond AML. The principles and techniques developed here could be applied to other cancers driven by specific genetic aberrations. As our understanding of the genetic underpinnings of various cancers deepens, such targeted gene editing strategies could become a cornerstone of cancer therapy. However, the journey from bench to bedside is fraught with challenges. While the in vitro and in vivo data are promising, translating these findings into a clinically viable treatment will require extensive further research. Key considerations include optimizing the delivery mechanisms for CRISPR-Cas9 components in humans, ensuring the specificity and safety of the treatment, and determining the long-term outcomes and potential side effects. Additionally, the ethical implications of gene editing in humans must be carefully considered. In conclusion, the study led by Professor Holm and his team represents a breakthrough in the use of CRISPR-Cas9 for cancer treatment. By demonstrating the feasibility and efficacy of a dual intron-targeting strategy in AML t(8;21), they have opened the door to a new era of precision medicine in oncology. This approach has the potential to improve outcomes for patients with AML and possibly other cancers, offering hope for more effective and less toxic treatments. The journey ahead is complex and requires careful navigation, but the path forward is undoubtedly promising.

One of the most exciting frontiers in medicine is the use of living cells as therapies. Using this approach to treat cancer, for example, many patients have been cured of previously untreatable disease. These advances employ the approaches of synthetic biology, a growing field that blends tools and concepts from biology and engineering. The new Northwestern technology uses computational modeling to more efficiently identify useful genetic designs before building them in the lab. Faced with myriad possibilities, modeling points researchers to designs that offer real opportunity.

Northwestern University scientists led by Dr. Josh Leonard together with Dr. Neda Bagheri from the University of Washington built a new cell therapies by engineering cells that can activate immune system. To engineer a cell, the authors first encoded the desired biological functions in a piece of DNA, and that DNA program is then delivered to a human cell to guide its execution of the desired function, such as activating a gene only in response to certain signals in the cell’s environment. Dr. Josh Leonard is an associate professor of chemical and biological engineering in the McCormick School of Engineering and a leading faculty member within Northwestern’s Center for Synthetic Biology. His lab is focused on using this kind of programming capability to build therapies such as engineered cells that activate the immune system, to treat cancer. Bagheri is an associate professor of biology and chemical engineering and a Washington Research Foundation Investigator at the University of Washington Seattle. Her lab uses computational models to better understand—and subsequently control—cell decisions.

The study, in which dozens of genetic circuits were designed and tested, is published in the journal Science Advances. Like other synthetic biology technologies, a key feature of this approach is that it is intended to be readily adopted by other bioengineering groups.

To date, it remains difficult and time-consuming to develop genetic programs when relying upon trial and error. It is also challenging to implement biological functions beyond relatively simple ones. The research team used a “toolkit” of genetic parts invented in Leonard’s lab and paired these parts with computational tools for simulating many potential genetic programs before conducting experiments. They found that a wide variety of genetic programs, each of which carries out a desired and useful function in a human cell, can be constructed such that each program works as predicted. Not only that, but the designs worked the first time.

The genetic circuits developed and implemented in this study are also more complex than the previous state of the art. This advance creates the opportunity to engineer cells to perform more sophisticated functions and to make therapies safer and more effective. With this new capability, we have taken a big step in being able to truly engineer biology.

Hearing is an exquisitely complex sense, and while hearing aids and cochlear implants are available to persons with hearing loss, these options do NOT restore normal hearing. As a consequence, there is a major push to develop biotherapies to restore normal auditory function. Among these therapies, gene therapy is the most promising. Within the ear and central to hearing are outer and inner hair cells (OHCs and IHCs). OHCs amplify sound signals while IHCs convert the mechanical information carried by sound waves into electrical signals that are transmitted to the brain. Many forms of genetic hearing loss have their origin in defects in genes in IHCs and OHCs that encode proteins essential to normal hair cell function.

Adeno-associated virus (AAV) is widely used in gene therapy due to its low pathogenicity and sustained expression in transduced cells. Its production is also simple, cheap, and fast. To date, the US Food and Drug Administration has approved AAV-based gene therapies for the treatment of spinal muscular dystrophy and Leber’s congenital amaurosis. AAVs are also being developed for therapeutic applications in the ear. To be successful, the tropism of AAVs for IHCs and OHCs must be defined. Some AAV serotypes, for example AAV1, AAV2, AAV8, AAV9 and Anc80, transduce IHCs and OHCs with different degrees of efficiency. For AAV in the ear to be successful, this variability must be clearly understood. In addition, because of the limited capacity of AAV to carry large genes (AAVs have a small viral loading capacity of no more than 5.0 kb), the ability of a single IHC or OHC to take in two different viruses (so called dual transduction) must be defined. Dual transduction enables delivery of larger transgenes by utilizing the intrinsic property of AAV to undergo concatamerization when separate vectors are co-transduced into a single cell.

These questions were addressed by scientists at University of Iowa – Dr. Ryotaro Omichi, Hidekane Dr. Yoshimura, Dr. Seiji Shibata and Professor Richard J.H. Smith – together with Professor Luk Vandenberghe at Harvard Medical School. Using a surgical approach they developed that involved the combination of round window membrane injection and canal fenestration (RWM+CF), they studied both single and dual AAV transduction in mature mice. The work is published in the Journal Molecular Therapy, Methods & Clinical Development.

In these pre-clinical experiments, the research team selected five AAV serotypes to study, namely AAV1, AAV2, AAV8, AAV9 and Anc80, and injected them into the cochlea. In single AAV experiments, they found that the transduction rate of IHCs and OHCs was highest with AAV2 (IHCs, 97%; OHCs, 84%). In addition, with the exception of AAV8, injection of single AAV serotypes did not alter auditory function. Based on these results, dual vector combinations of AAV2-eGFP and AAV2-mCherry (AAV2-2), AAV2-eGFP and AAV9-mCherry (AAV2-9), and AAV9-eGFP and AAV9-mCherry (AAV9-9) were tested. AAV2-2 dual transduction was found to be as robust and efficient as that of the single AAV2.

Their data indicate that co-transduction is not influenced by cooperation between the two vectors or by preferential selection of the hair cells, which is relevant because dual vector transduction was believed to be inferior to single vector transduction based on ocular and retinal studies. For example, the retinal transduction efficiency of dual AAV8 hybrid vectors has been reported to be 6% and 40% of the transduction rate with a single AAV8 vector in both mice and pigs.  While this study observed a similar trend with dual AAV9-9 co-transduction, for AAV2-2 and AAV2-9, co-transduction rates were similar to single vector transduction rates. These findings suggest that specific combinations of dual vectors are likely to be important, an encouraging and important observation as experiments with AAV continue to explore its suitability as the viral vector of choice in the treatment of human deafness with gene therapy.

Adeno-associated viruses (AAVs) have become promising vehicles for delivering gene therapies to defective tissues in the human body because they are non-pathogenic and can transfer therapeutic DNA into target cells. However, while the first gene therapy products approved by the Federal Drug Administration (FDA) use AAV vectors and others are likely to follow, AAV vectors still have not reached their full potential to meet gene therapeutic challenges.

First, currently used AAV capsids—the spherical protein structures enveloping the virus’ single-stranded DNA genome which can be modified to encode therapeutic genes—are limited in their ability to specifically hone in on the tissue affected by a disease and their wider distribution throughout the human body causes them to be diluted. And secondly, patients’ immune systems, after having been exposed to a similar AAV virus, can produce neutralizing antibodies that, even at low levels, can destroy AAVs upon re-exposure (neutralization), blocking the delivery of their therapeutic DNA payloads.

To overcome this neutralization problem, researchers are engineering enhanced AAV capsids they hope to be able to evade the immune system. Currently used methods, including “directed evolution” strategies that fast-track the evolution of a protein in laboratory conditions, only can create a limited diversity of capsids with most of them still resembling the naturally occurring AAV variants known as serotypes. However, it remains difficult to generate sufficient diversity using this approach without losing other desired functions of the capsid, such as their stability or ability to bind to specific cell types.

Now, a new study initiated by Wyss Core Faculty member George Church’s Synthetic Biology team at Harvard’s Wyss Institute for Biologically Inspired Engineering, and driven by a collaboration with Google Research has applied a computational deep learning approach to design highly diverse capsid variants from the AAV2 serotype across DNA sequences encoding a key protein segment that plays a role in immune-recognition as well as infection of target tissues. AAV2 is the most-studied serotype and has been used in the first FDA approved gene therapy, to treat a blinding disease.

Starting from a relatively small collection of capsid data, the team trained multiple machine learning methods and used them to design 200,000 virus variants. 110,689 of these variants produced viable AAV viruses. Between any two naturally occurring AAV serotypes, 12 amino acids within this segment are expected to differ. The team’s effort produced more than 57,000 variants that exhibited much higher diversity than this, some containing up to 29 combined substituted or additionally inserted amino acids. The findings are published in Nature Biotechnology.

“Our approach achieves the highest functional diversity of any capsid library thus far. It unlocks vast areas of functional but previously unreachable sequence space, with many potential applications for generating improved viral vectors, like AAVs with much reduced immunogenicity and much improved target tissue selectivity, and also for highly efficient gene therapies,” said last-author Eric Kelsic, Ph.D., who started the project with Church, Ph.D., and co-founded the startup Dyno Therapeutics where he is now CEO. Dyno Therapeutics’ mission is to develop advanced gene therapy delivery vehicles by employing cutting-edge artificial intelligence (AI) approaches.

Using multiple design strategies, the team first generated smaller data sets on which they could train several machine learning models. These were collections of AAV capsids with variable numbers of mutations introduced in a 28 amino acid segment of the AAV2 VP3 protein that forms part of the capsid and exposes it to neutralizing antibodies. A high-throughput method enabling the synthesis of mutated capsid sequences and in vitro experiments for testing which ones efficiency produced viable stable capsids, provided a highly effective test bed for their overall approach. The results from this first experimental study then were used by the team as training data for three alternative machine learning models that generated much larger numbers of diverse capsid variants to be tested with a final validation experiment.

A central bottleneck in the creation of diverse AAV capsids and variants that can evade neutralization is the production of capsids that remain stable: most of the variants will fail to assemble into functional capsids or package their AAV genomes. “The deep neural network models that we deployed with our Google collaborators accurately predicted capsid viability across extremely diverse variants. Reaching this level of diversity in the capsid segment is an important milestone that we can build on to find immune-evading capsids for gene therapy,” said co-first author Sam Sinai, Ph.D., a former graduate student of Church who joined Kelsic’s team at the Wyss Institute and is a co-founder leading the machine learning team at Dyno Therapeutics. “And we can take similar approaches to create AAV capsids with much improved tissue selectivity.”

In 2019, a former Wyss team including Kelsic, Sinai, and their mentor Church published a related approach in Science in which they mutated one by one each of the 735 amino acids within the entire AAV2 capsid in different ways. What they called a “wide” search resulted in a large AAV library that identified changes affecting AAV2’s viability and its “homing” potential to specific organs in mice, as well as a previously unknown accessory protein that binds to cell membranes and which was hidden within the capsid-encoding DNA sequence. In their previous study, the researchers used a simple experimental model to optimize the tissue targeting ability of the virus.

This new study involving machine learning models developed with Google Research nicely complements our earlier work in that it focuses on a small, but very important, region of the AAV capsid with an unprecedented resolution. It shows that neural networks combined with the high-throughput synthetic testing developed is changing the way gene delivery vehicles and protein drugs are designed.

The study gives a glimpse into the future as artificial intelligence approaches, such as machine learning, are opening up vast new design spaces that enable the development of entirely new drugs and drug delivery approaches for combating innumerable challenges to human health

Pancreatic cancer occurs within the tissues of the pancreas, which is a vital endocrine organ located behind the stomach. The pancreas plays an essential role in digestion by producing enzymes that the body needs to digest fats, carbohydrates, and proteins. pancreatic cancer makes up about 3 percent of cancer diagnoses in the United States and 7 percent of cancer deaths

A new mouse study by researchers led by Angus Cameron, PhD, senior lecturer at Barts Cancer Institute at Queen Mary University of London, reveals new insight into how healthy cells help pancreatic tumors develop, which may pave the way for the development of new drugs for pancreatic cancer. The researchers discovered that blocking the expression of a protein, called PKN2, changed the behavior of healthy cells around the tumor called fibroblasts. Their findings suggest targeting fibroblasts may change their behavior and affect how pancreatic cancer develops. When the researchers blocked expression of PKN2 in the healthy cells of a preclinical model of pancreatic cancer, the tumor grew more aggressively. Their findings are published in Cell Reports.

The authors found that PKN2 regulates both the activation of mouse PSCs and mouse embryonic fibroblasts (MEFs) into myofibroblasts. They also identified PKN2 as a novel regulator of the mechano-sensor YAP, which is central to myofibroblast function. Intriguingly, loss of PKN2 in PSCs resulted in a switch in cellular invasive mechanism in heterotypic spheroid cultures, suppressing PSC invasion while promoting polarized epithelial outgrowth. Further, stromal deletion of PKN2 in vivo results in more locally invasive tumors, with accompanying pro-invasive changes to the matrisome signature. According to the authors, preventing myofibroblast differentiation in malignancy may therefore limit the tumor-suppressive role of fibroblasts, counter to the dogma that CAFs support cancer invasion. Their work also highlights the potential impact that targeting specific fibroblast phenotypes may have on functionally distinct CAF subtypes in PDAC.

“Fibroblasts are like the gatekeepers of pancreatic cancer tumors, and fibroblasts may have both positive and negative roles to play in cancer progression.

The authors found that, when activated through PKN2, fibroblasts can actually act as a defense mechanism to limit cancer spread by keeping the cancer cells tightly compacted within the tumor. Blocking PKN2 suppresses the ability of fibroblasts to contain the cancer cells; however, it also means that they may let more immune cells into the tumor. This novel finding could have broad implications for how we target stromal fibroblasts to treat cancer.

Together, the authors identified PKN2 as a potential target to modulate the pathological activation of fibroblasts. However, preventing fibroblast activation could also suppress the ability of myofibroblasts to contain and suppress malignant tumor growth by altering the fibroblast matrisome and secretome. The fibrotic, hypovascular nature of the pancreatic cancer stroma nonetheless remains a critical barrier to both chemo- and immunotherapy. Targeting fibrosis to improve therapy responses while retaining the tumor-suppressive functions of fibroblasts thus presents a clinical dilemma.

The researchers are now studying the altered profile of immune cells within pancreatic cancer tumors. To improve the outcomes for patients, the research team aim to identify new strategies to target cancer cells as well as the normal cells supporting cancer growth, and find ways to help the body’s immune system fight back against cancer. Their study contributes to the understanding of the biology of the invasive process in pancreatic cancer, and the roles that fibroblasts play.  Future work will aim to identify effective drugs to target PKN2, which can be used in laboratory models of pancreatic and other cancers.