Periodic Reporting for period 4 - ImmunoStem (Dissecting and Overcoming Innate Immune Barriers for Therapeutically Efficient Hematopoietic Stem Cell Gene Engineering)
Reporting period: 2023-10-01 to 2025-02-28
The low gene manipulation efficiency of human hematopoietic stem cells (HSC) remains a major hurdle for sustainable and broad clinical application of innovative therapies for a wide range of disorders. Indeed, high vector doses and prolonged ex vivo culture are still required for clinically relevant levels of gene transfer even with the most established lentiviral vector-based delivery platforms.
Current and emerging gene transfer and editing technologies expose HSC to components potentially recognized by host antiviral factors and nucleic acid sensors that likely restrict their genetic engineering and contribute to broad individual variability in clinical outcomes observed in recent gene therapy trials. Nevertheless, specific effectors are yet to be identified in HSC. We have recently identified an antiviral factor that potently blocks gene transfer in HSC and have discovered small molecules that efficiently counteract it. This is the first example of how manipulating a single host factor can significantly impact gene transfer efficiencies in HSC but likely represents the mere tip of the iceberg of the plethora of innate sensing mechanisms potentially hampering genetic manipulation of this primitive cell compartment.
On these premises, this proposal has aimed to determine the restriction factors and innate sensing pathways that prevent efficient modification of HSC and to mitigate their effect using methods developed through a thorough understanding of their mechanisms of action. The following Specific Aims have been pursued:
Aim 1. Dissecting the determinants of intrinsic antiviral resistance in HSC.
Aim 2. Investigating HSC-specific nucleic acid sensing mechanisms.
Aim 3. Development of improved HSC gene engineering strategies.
Overall, this project has delivered significant molecular innate into pathogen recognition in gene therapy target tissues that will allow ground-breaking progress in the development of cutting-edge cell and gene therapies and to fight infectious and autoimmune diseases.
Current and emerging gene transfer and editing technologies expose HSC to components potentially recognized by host antiviral factors and nucleic acid sensors that likely restrict their genetic engineering and contribute to broad individual variability in clinical outcomes observed in recent gene therapy trials. Nevertheless, specific effectors are yet to be identified in HSC. We have recently identified an antiviral factor that potently blocks gene transfer in HSC and have discovered small molecules that efficiently counteract it. This is the first example of how manipulating a single host factor can significantly impact gene transfer efficiencies in HSC but likely represents the mere tip of the iceberg of the plethora of innate sensing mechanisms potentially hampering genetic manipulation of this primitive cell compartment.
On these premises, this proposal has aimed to determine the restriction factors and innate sensing pathways that prevent efficient modification of HSC and to mitigate their effect using methods developed through a thorough understanding of their mechanisms of action. The following Specific Aims have been pursued:
Aim 1. Dissecting the determinants of intrinsic antiviral resistance in HSC.
Aim 2. Investigating HSC-specific nucleic acid sensing mechanisms.
Aim 3. Development of improved HSC gene engineering strategies.
Overall, this project has delivered significant molecular innate into pathogen recognition in gene therapy target tissues that will allow ground-breaking progress in the development of cutting-edge cell and gene therapies and to fight infectious and autoimmune diseases.
The overall goal of ImmunoStem has been to determine the restriction factors and innate sensing pathways that prevent efficient modification of HSC and to mitigate their effect using methods developed through a thorough understanding of their mechanisms of action. We have successfully implemented actions as follows:
• Aim 1. Dissecting the determinants of intrinsic antiviral resistance in HSC.
We have gained mechanistic insight into how innate immune factors block gene therapy vector entry into HSPC and how our pharmacological strategies counteract them. We have identified the cellular pathway involved in antiviral factor degradation by transduction enhancers and uncovered novel molecular determinants required for their antiviral activity not only against gene therapy vectors but also other pathogens, including the pandemic causing SARS-CoV2. In addition, our studies on enhancement of gene therapy in HSC have unravelled additional, potentially beneficial effects our transduction enhancers seem to have on pathways relevant for stem cell biology.
• Aim 2. Investigating HSC-specific nucleic acid sensing mechanisms.
We have taken advantage of viral vectors to dissect how HSPC detect incoming pathogens leading to activation of antiviral type I IFN responses. We have also investigated these responses in other gene therapy target tissues and diseased HSC.
• Aim 3. Development of improved HSC gene engineering strategies.
To reliably test enhanced transduction and gene editing protocols in pre-clinical in vivo studies, we have set-up and characterized a research-grade lentiviral vector (LV) purification pipeline. Taking advantage of this vector production pipeline, we have tested novel gene therapy protocols that not only enhance efficacy of genetic engineering but improve engraftment and clonality of edited HSPC in vivo. In addition, we have developed novel transduction protocols that enable efficient modification of quiescent, unmanipulated HSPCs while preserving their higher engraftment capacity and yielding more polyclonal output in vivo.
Overall, the ImmunoStem project has delivered substantial advancements to our understand of how and which innate immune barriers prevent efficient genetic manipulation of HSPC together with pharmacological approaches that can be developed towards clinical application in the future to efficiently transduce HSPC, including the most quiescent unstimulated cells.
• Aim 1. Dissecting the determinants of intrinsic antiviral resistance in HSC.
We have gained mechanistic insight into how innate immune factors block gene therapy vector entry into HSPC and how our pharmacological strategies counteract them. We have identified the cellular pathway involved in antiviral factor degradation by transduction enhancers and uncovered novel molecular determinants required for their antiviral activity not only against gene therapy vectors but also other pathogens, including the pandemic causing SARS-CoV2. In addition, our studies on enhancement of gene therapy in HSC have unravelled additional, potentially beneficial effects our transduction enhancers seem to have on pathways relevant for stem cell biology.
• Aim 2. Investigating HSC-specific nucleic acid sensing mechanisms.
We have taken advantage of viral vectors to dissect how HSPC detect incoming pathogens leading to activation of antiviral type I IFN responses. We have also investigated these responses in other gene therapy target tissues and diseased HSC.
• Aim 3. Development of improved HSC gene engineering strategies.
To reliably test enhanced transduction and gene editing protocols in pre-clinical in vivo studies, we have set-up and characterized a research-grade lentiviral vector (LV) purification pipeline. Taking advantage of this vector production pipeline, we have tested novel gene therapy protocols that not only enhance efficacy of genetic engineering but improve engraftment and clonality of edited HSPC in vivo. In addition, we have developed novel transduction protocols that enable efficient modification of quiescent, unmanipulated HSPCs while preserving their higher engraftment capacity and yielding more polyclonal output in vivo.
Overall, the ImmunoStem project has delivered substantial advancements to our understand of how and which innate immune barriers prevent efficient genetic manipulation of HSPC together with pharmacological approaches that can be developed towards clinical application in the future to efficiently transduce HSPC, including the most quiescent unstimulated cells.
During this project we have been able to uncover novel determinants of innate immunity and nucleic acid sensing in human cells, including hematopoietic stem cells.
Our studies of antiviral activity in the context of HSC gene therapy have revealed previously unknown molecular determinants involved in antiviral control by innate immune factors. These studies together with the historical times of the COVID-19 pandemic, have also allowed us to explore the role of these factors also in the context of the pandemic causing SARS-CoV2 infection. Together, these studies provide significant insight into how these antiviral factors impact gene therapy and viral pathogenesis, paving the way for the development of novel strategies to either overcome or enhance this restriction as needed.
In association with the studies on these antiviral mechanisms of actions we are understanding how we can counteract these antiviral factors in the context of enhanced gene therapies. These studies inform the development of novel, easier to produce, enhancers of gene therapy and provide insight into how antiviral responses are regulated in human stem cells. In addition, our studies on pharmacological enhancement of gene therapy in HSC is uncovering additional effects our compound seems to have on pathways relevant for stem cell biology. We predict these findings may have particular relevance in the context of diseased HSC that could benefit from the transduction enhancer not only for enhanced gene transfer but also in terms of fitness. Similarly, other blood cell types that are attractive targets for advanced immunotherapies such as CAR-T cells will potentially benefit from the use of such enhancers as their genetic manipulation remains challenging and prolonged ex vivo culture leads to exhaustion.
Importantly, we have discovered a novel, still cryptic, innate sensor of retroviruses that detects incoming viral particles in HSC and primary macrophages, leading to robust activation of type I IFN responses. Much of the focus in the field of innate immunity against viral infections has been directed towards sensing of nucleic acids such as RNA and DNA upon viral infections. Structural recognition of incoming virions has been shown to occur for HIV but very little knowledge on potential recognition of other retroviral particles is available. Identifying novel host factors of the innate sensing machinery wired towards structural components of viral pathogens can have potentially broad acting implications for the development not only of enhanced gene and antiviral therapies but also to harness these responses immunostimulatory applications in the context of antitumoral approaches.
Our work has also enabled us to contribute to the understanding of how these innate immune sensing pathways can contribute to other autoimmune pathologies beyond the monogenic blood disorders that can be treated with gene therapies.
Our studies of antiviral activity in the context of HSC gene therapy have revealed previously unknown molecular determinants involved in antiviral control by innate immune factors. These studies together with the historical times of the COVID-19 pandemic, have also allowed us to explore the role of these factors also in the context of the pandemic causing SARS-CoV2 infection. Together, these studies provide significant insight into how these antiviral factors impact gene therapy and viral pathogenesis, paving the way for the development of novel strategies to either overcome or enhance this restriction as needed.
In association with the studies on these antiviral mechanisms of actions we are understanding how we can counteract these antiviral factors in the context of enhanced gene therapies. These studies inform the development of novel, easier to produce, enhancers of gene therapy and provide insight into how antiviral responses are regulated in human stem cells. In addition, our studies on pharmacological enhancement of gene therapy in HSC is uncovering additional effects our compound seems to have on pathways relevant for stem cell biology. We predict these findings may have particular relevance in the context of diseased HSC that could benefit from the transduction enhancer not only for enhanced gene transfer but also in terms of fitness. Similarly, other blood cell types that are attractive targets for advanced immunotherapies such as CAR-T cells will potentially benefit from the use of such enhancers as their genetic manipulation remains challenging and prolonged ex vivo culture leads to exhaustion.
Importantly, we have discovered a novel, still cryptic, innate sensor of retroviruses that detects incoming viral particles in HSC and primary macrophages, leading to robust activation of type I IFN responses. Much of the focus in the field of innate immunity against viral infections has been directed towards sensing of nucleic acids such as RNA and DNA upon viral infections. Structural recognition of incoming virions has been shown to occur for HIV but very little knowledge on potential recognition of other retroviral particles is available. Identifying novel host factors of the innate sensing machinery wired towards structural components of viral pathogens can have potentially broad acting implications for the development not only of enhanced gene and antiviral therapies but also to harness these responses immunostimulatory applications in the context of antitumoral approaches.
Our work has also enabled us to contribute to the understanding of how these innate immune sensing pathways can contribute to other autoimmune pathologies beyond the monogenic blood disorders that can be treated with gene therapies.