Document text
Principal Investigator: Andre LaRochelle
Organization: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
Fiscal Year: 2024
Award: $1,497,052
Funding agency: National Heart Lung and Blood Institute
1. Addressing the Cellular Scarcity in FA
1.1 Eltrombopag to Enhance Marrow HSPCs. We have identified eltrombopag (EPAG), a small molecule agonist of the thrombopoietin receptor c-MPL, as a transformative therapeutic approach to augment the availability of HSPCs in FA patients. EPAG enhances DNA repair and reduces HSPC hypersensitivity to proinflammatory cytokines, increasing their resilience. In a phase II trial (NCT03204188) involving FA-associated BMF, 50% of the cohort demonstrated a 14-fold increase in bone marrow CD34+ HSPCs. These findings suggest that EPAG could expand the pool of FA patients eligible for gene therapy. Detailed exploration of this project is provided in a separate section of the Annual Report.
2. Develop DSB-Free Gene Editing Strategies in HSPCs
2.1 Base Editing for Point Mutation Correction. Adenine base editing (ABE) offers a strategy for correcting point mutations without inducing DSBs. We are targeting the FANCA c.2638C>T, p.Arg880* point mutation identified in a subset of FA patients. Initial optimization are conducted using EBV-transformed cell lines derived from PBMNCs obtained from study participants before application in FA HSPCs.
2.2 Transposase-Mediated Gene Editing for Targeted Integration. We have developed a dCas9-Tn5 fusion protein to enable targeted gene insertion without unremediated DSBs. Proof-of-concept studies have shown successful integration events at the AAVS1 locus, and we have further enhanced this approach by engineering dimers and oligomers of Tn5 for improved gene insertion efficiency. The effectiveness of these modifications will be quantified in human cells using HTS.
3. Facilitate Safe and Efficient Engraftment of Gene-Edited HSPCs for Ex Vivo Gene Therapy
3.1 Pre-Transplant Conditioning with Chemotherapy-Free Regimens. We hypothesized that cMPL might be a relevant antigen for an antibody-based targeted depletion of human HSPCs and provide the basis for a safer conditioning regimen prior to transplant. To investigate this possibility, we have produced a recombinant anti-cMPL bivalent (bi) single-chain fragment variable (scFV) fused with diphtheria toxin (DT) truncated at residue 390 (DT390-biscFV(cMPL)). This agent has demonstrated HSPC depletion in preclinical models, with further dosage optimization underway [BioRxiv 2024: doi: 10.1101/2024.02.24.581887].
3.2 Increase Cell Dose by Ex Vivo Expansion of Gene-edited HSPCs. We are developing a synthetic, cytokine-free culture system for ex vivo expansion of genetically modified HSPCs. Our strategy includes promoting HSPC self-renewal through LNP-mediated transient expression of transcription factors, suppressing differentiation using CD38 inhibitors, and mitigating ER stress by evaluating hypoxic conditions and synthetic Hsf1 agonists.
3.3 Increase Cell Fitness by Overcoming Innate Immune Responses. A growing body of experimental evidence suggests a pivotal role of host antiviral factors and nucleic acid sensors in limiting the efficacy of HSPC genetic manipulation. To characterize innate immune pathways triggered by reagents used for genetic engineering of HSPCs, we are conducting unbiased proteomic and single-cell transcriptomic screens on human HSC-enriched populations exposed to commonly used gene delivery systems, including vectors based on RV, LV, FV and AAV6, as well electroporation and LNP carriers of nucleic acid constituents (e.g., DNA and mRNA). These findings will inform novel approaches to overcome immune blocks to nucleic acid delivery and enhance gene correction efficiencies by promoting cellular survival and fitness.
3.4 Evaluate the Impact of Post-Transplant G-CSF Administration on Gene-Edited HSPCs. Granulocyte colony stimulating factor (G-CSF) is commonly used to accelerate recovery from neutropenia following chemotherapy and autologous transplantation of HSPCs for malignant disorders. However, its utility after ex vivo gene therapy in human HSPCs remains unexplored. We showed that administering G-CSF from day 1 to 14 post-transplant impedes engraftment of CRISPR-Cas9 gene-edited human HSPCs in murine xenograft models. G-CSF affects gene-edited HSPCs through a cell-intrinsic mechanism, causing proliferative stress and amplifying the early p53-mediated DNA damage response triggered by Cas9-mediated DNA DSBs. This underscores a threshold mechanism where p53 activation must reach a critical level to impair cellular functions. Transiently inhibiting p53 or delaying the initiation of G-CSF treatment to day 5 post-transplant attenuates its negative impact on gene-edited HSPCs. The potential for increased HSPC toxicity associated with post-transplant G-CSF administration in CRISPR-Cas9 autologous HSPC gene therapy warrants consideration in clinical trials [BioRxiv 2023: doi: 10.1101/2023.06.29.547089].
4. Develop in Vivo Gene Therapy Strategies
4.1 Development and Evaluation of novel LNP Formulations. We have re-engineered standard LNP formulations with a novel ionizable amino dendrimer lipid (4A3-SC8), and demonstrated up to 34% indel formation in human HSPCs in vitro. For in vivo biodistribution analyses, we encapsulated these LNPs with sodium-iodide symporter (NIS) reporter mRNA and demonstrated specific delivery to hematopoietic organs in non-human primates (NHPs) post-infusion. Furthermore, we conducted proof-of-concept CRISPR/Cas9-mediated gene editing in NHPs by encapsulating LNPs with Cas9 mRNA and sgRNA targeting the CCR5 locus. TIDE analysis revealed evidence of gene editing in peripheral blood (PB) and BM MNCs up to 14 days post-infusion, although indel rates were low (<3%), underscoring the need for further optimization.
4.2 Enhance LNP Delivery Systems for Safe and Efficient HSPC Gene Editing in Vivo
4.2.1 Overcoming Mononuclear Phagocyte System (MPS) Capture of LNPs. The capture of LNPs by the MPS presents a significant obstacle to efficient targeting of HSPCs. To address this challenge, we are developing chemically-conjugated LNPs with a recombinant anti-cMPL bivalent scFV and pre-treat recipient NHPs with MPS blockade agents (e.g., dextran sulfate, colchicine, liposomal clodronate). Initial evaluation of cMPL-conjugated LNPs involves in vitro binding assays (e.g., flow cytometry, ELISA, SPR), internalization assays (e.g., confocal microscopy), and functional assays (e.g., CFU cultures, xenotransplantation).
4.2.2 Enhancing Endosomal Release. Suboptimal release of LNP payloads from endosomes into the cytosol and nucleus of HSPCs hinders effective in vivo gene editing. To overcome this, we substituted the original dendrimer lipid 4A3-SC8 with 4A3-SCC-PH, featuring novel disulfide bond-bridged ester linkers and branched hydrophobic tails to improve endosomal release. Additionally, we are exploring endosomal escape agents, such as chloroquine, to further enhance cargo release. Efficiency of endosomal release is evaluated in vitro using a FRET-based approach, hemolysis assays, and colocalization studies via confocal microscopy.
4.3 Evaluation in NHPs. LNP formulations optimized for HSPC targeting (§4.2.1) and endosomal release (§4.2.2) will be encapsulated with Cas9 mRNA/sgRNA targeting the CCR5 reporter gene, base editors (§2.1) or payloads for targeted gene insertion (§2.2), and infused into NHPs pre-treated with MPS blockade agents. Safety and efficacy will be monitored longitudinally by HTS, WGS, integration site analysis and clinical monitoring.
Terms: <1H-Purin-6-amine><Acceleration><Address><Adenine><Affect><Agonist><Allergy><Annual Reports><Antibodies><Antigens><Antioncogene Protein p53><Assay><Attenuated><Autograft><Autologous><Autologous Transplantation><Autotransplant><Basal Transcription Factor><Basal transcription factor genes><Basic Research><Basic Science><Binding><Bioassay><Biodistribution><Biological Assay><Blood Diseases><Blood Precursor Cell><Bone Marrow><Bone Marrow Reticuloendothelial System><Bone marrow failure><Burkitt Herpesvirus><Burkitt Lymphoma Virus><C-C CKR-5><C-C CKR-5 Gene><C-C Chemokine Receptor Type 5><C-C Chemokine Receptor Type 5 Gene><CC Chemokine Receptor 5><CC-CKR-5><CC-CKR-5 Gene><CC-CKR5><CCCKR5><CCCKR5 Gene><CCR-5><CCR-5 Gene><CCR5><CCR5 Protein><CCR5 Receptors><CCR5 gene><CD195 Antigen><CD195 Antigen Gene><CD34><CD34 gene><CHEMR13><CHEMR13 Gene><CKR-5><CKR-5 Gene><CKR5><CKR5 Gene><CKR5 Receptors><CMKBR5><CMKBR5 Gene><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancers><Cas nuclease technology><Cell Body><Cell Function><Cell Nucleus><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular Tumor Antigen P53><Chemokine (C-C Motif) Receptor 5><Chemokine (C-C) Receptor 5><Chemokine (C-C) Receptor 5 Gene><Chimera Protein><Chimeric Proteins><Chlorochin><Chloroquine><Clinical><Clinical Trials><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Colchicine><Colony Stimulating Factor 3><Confocal Microscopy><Congenital Pancytopenia><Corynebacterium Diphtheriae Toxin><Cytosol><DNA><DNA Damage><DNA Damage Repair><DNA Double Strand Break><DNA Injury><DNA Repair><DNA Therapy><Defect><Dendrimers><Dendritic Compounds><Dendrons><Deoxyribonucleic Acid><Development><Dextran Sulfate><Dichloromethylene Diphosphonate><Diphtheria Toxin><Disease><Disorder><Dose><Double Strand Break Repair><EB virus><EBV><ELISA><ER stress><Effectiveness><Electroporation><Eligibility><Eligibility Determination><Encapsulated><Endosomes><Engineering><Engraftment><Ensure><Enzyme-Linked Immunosorbent Assay><Epstein Barr Virus><Esters><Evaluation><Event><Exposure to><FANCA><FANCA Protein><FRET><Face><Fanconi Anemia><Fanconi Anemia Complementation Group A Protein><Fanconi Anemia Group A Complementing Protein><Fanconi Anemia Group A Protein><Fanconi Panmyelopathy><Fanconi dysplasia><Fanconi's Anemia><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Fluorescence Resonance Energy Transfer><Formulation><Fusion Protein><Förster Resonance Energy Transfer><Gene Delivery><Gene Modified><Gene Transfer Clinical><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Engineering><Genetic Engineering Biotechnology><Genetic Engineering Molecular Biology><Genetic Intervention><Granulocyte Colony-Stimulating Factor><HHV-4><HHV4><HIV-1 Fusion Co-Receptor><HIV-1 Fusion Co-Receptor Gene><HPCA1><Harvest><Hematologic Body System><Hematologic Diseases><Hematologic Organ System><Hematological Disease><Hematological Disorder><Hematopoietic><Hematopoietic Body System><Hematopoietic Progenitor Cells><Hematopoietic System><Hematopoietic stem cells><Hemolysis><Hereditary><Heterograft><Heterologous Transplantation><Human><Human Herpesvirus 4><Hydrophobicity><Hypersensitivity><Hypoxia><Hypoxic><Immune><Immunes><Impairment><In Vitro><Individual><Infectious Mononucleosis Virus><Inflammation><Inflammation Mediators><Inflammatory><Infusion><Infusion procedures><Inherited><Inherited bone marrow failure><Innate Immune Response><Investigation><Khingamin><Lipids><Liposomal><Liposomes><MPL gene><MPLV><Malignant><Malignant - descriptor><Malignant Neoplasms><Malignant Tumor><Marrow><Mediating><Messenger RNA><Methods><Mice><Mice Mammals><Modern Man><Modification><Molecular Interaction><Monitor><Mononuclear><Mouse Homolog of Myeloproliferative Leukemia Virus><Murine><Mus><Myeloproliferative Leukemia Virus Oncogene><NIS Gene><NIS protein><Neutropenia><Non-Viral Vector><Nucleic Acids><Nucleus><Oncoprotein p53><Organ><Oxygen Deficiency><P50 Mechanism><P50 Program><P53><Pancytopenia><Participant><Patients><Phagocytes><Phagocytic Cell><Phosphoprotein P53><Phosphoprotein pp53><Pluripoietin><Point Mutation><Population><Pre-Clinical Model><Preclinical Models><Primary Erythroid Hypoplasia><Process><Protein TP53><Proteomics><Protocol><Protocol Screening><Protocols documentation><Reagent><Receptosomes><Recombinant DNA Technology><Recombinants><Recovery><Regenerative Medicine><Regimen><Reporter><Reporter Genes><Research><Risk><Role><SLC5A5><SLC5A5 gene><Safety><Solute Carrier Family 5 (Sodium Iodide Symporter), Member 5><Specialized Center><Stress><Subcellular Process><System><TP53><TP53 gene><TPOR><TRP53><Tail><Therapeutic><Thrombopoietin Receptor><Toxic effect><Toxicities><Transcription Factor Proto-Oncogene><Transcription factor genes><Transformed Cell Line><Translating><Translational Research><Translational Science><Transplantation><Transplantation Conditioning><Transposase><Treatment Factor><Tumor Protein p53><Tumor Protein p53 Gene><Unscheduled DNA Synthesis><Viral><Viral Vector><Vitamin B4><Xenograft><Xenograft Model><Xenograft procedure><Xenotransplantation><amebocyte><attenuate><attenuates><autologous graft><autotransplantation><base editing><base editor><blood cell progenitor><blood disorder><blood progenitor><blood stem cell><blood-forming stem cell><bone marrow failure syndrome><chemical conjugate><chemotherapy><clinical efficacy><clinical practice><clodronate><cohort><conditioning><congenital aplastic anemia><cost><cytokine><design><designing><developmental><dimer><disulfide bond><dosage><effective therapy><effective treatment><electroporative delivery><endoplasmic reticulum stress><enzyme linked immunoassay><erythrolysis><faces><facial><fitness><flow cytophotometry><gene corrected><gene correction><gene delivery system><gene editing method><gene editing methodology><gene editing strategy><gene editing techniques><gene electrotransfer><gene manipulation><gene modification><gene repair therapy><gene therapy><gene-based therapy><gene-editing approach><genetic manipulation><genetic therapy><genetically engineered><genetically manipulate><genetically modified><genetically perturb><genomic correction><genomic therapy><granulocyte colony stimulating factor><hematopoietic progenitor><hematopoietic stem progenitor cell><hemopoietic><hemopoietic progenitor><hemopoietic stem cell><hydrogen sulfate Dextran><immunogen><immunogenicity><improved><in vivo><indel><inflammatory mediator><infusions><inherited disease of bone marrow failure><inhibitor><innate immune pathways><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><insight><integration site><lipid based nanoparticle><lipid nanoparticle><mRNA><malignancy><manufacture><mutation correction><nano particle delivery><nanoparticle delivered><nanoparticle delivery><neoplasm/cancer><new approaches><non-human primate><nonhuman primate><nonviral vector><novel><novel approaches><novel strategies><novel strategy><nucleic acid delivery><p53 Antigen><p53 Genes><p53 Tumor Suppressor><peripheral blood><phase 2 trial><phase II trial><post-transplant><post-transplantation><posttransplant><posttransplantation><programs><protein p53><resilience><resilient><response><self-renew><self-renewal><sensor><small molecule><social role><sodium-iodide symporter><tool><transcription factor><transcriptomics><translation research><translational investigation><transplant><vector><xeno-transplant><xeno-transplantation><xenograft transplant model><xenotransplant model>