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Principal Investigator: Sheng Tong
Organization: UNIVERSITY OF KENTUCKY
Fiscal Year: 2023
Award: $229,500
Funding agency: National Heart Lung and Blood Institute
Summary
CRISPR/cas9 gene editing has shown great promise for the treatment of genetic hematologic
disorders including sickle cell disease and β-thalassemia. Current therapeutic strategies are
primarily focused on ex vivo gene editing of autologous patient-derived hematopoietic
stem/progenitor cells (HSPCs), which require isolation of patients’ HSPCs, ex vivo gene editing,
selection and expansion of corrected HSPCs, and transplantation back into the patients.
Despite its initial success, the clinical translation of this technique is hampered by the difficulties
in ex vivo processing of HSPCs, the risks associated with myeloablation, the low engraftment
efficiency, and the prohibitively high cost of individualized cell therapy. Recent studies have
shown that HSPCs are sustained in specialized niches in the adult bone marrow. HSPC niches
are located near the sinusoidal blood vessels, where the fenestrated endothelium is highly
permeable to nanoparticles and viral vectors. To this end, we propose that the HSPCs in the
bone marrow can be gene-edited by CRISPR/cas9 in situ. However, in vivo CRISPR/cas9 gene
editing can have substantial off-target effects due to the systemic dissemination of the delivery
vehicles and the non-specific activities of the cas9 nuclease. Recently, we developed a novel
gene-editing platform that combines the baculoviral vector with magnetic nanoparticles (MNP-
BV). Compared with conventional viral vectors, the baculoviral vector can transduce a broad
range of mammalian cells without replication. MNP-BV uses an external magnetic field and the
intrinsic complement system as the on- and off-switch for site-specific transgene delivery. In this
project, we will develop an MNP-BV-based gene-editing technique for precise gene editing of
HSPCs in the bone marrow. MNP-BV will be administrated via intraosseous infusion. We will
design a magnetic targeting method to enhance the retention of MNP-BV in the bone marrow
and the extravasation of MNP-BV to the perisinusoidal niches. Furthermore, the baculoviral
vector has a large DNA loading capacity (>38 kb) and thus can deliver inducible cas9 or gRNA
expression cassettes targeting specific cell populations. We will design gRNAs that can only be
activated by microRNAs (miRNAs) highly expressed in HSPCs. The central hypothesis is that
by combining intraosseous infusion, magnetic targeting, and miRNA-mediated posttranslational
regulation, the MNP-BV system can efficiently and precisely transduce HSPCs in the bone
marrow and correct hematological diseases-associated gene mutations. The success of this
project will pave the way for developing an effective and low-cost cure for a range of
hematological diseases.
Terms: <21+ years old><Adult><Adult Human><Allogenic><Autograft><Autologous><Autologous Transplantation><Autotransplant><B-globin><B-thalassemia><Back><Basic Research><Basic Science><Binding Sites><Blood Diseases><Blood Precursor Cell><Blood Vessels><Bone Marrow><Bone Marrow Blood-Deriving Cell><Bone Marrow Blood-Forming Cell><Bone Marrow Cells><Bone Marrow Purging><Bone Marrow Reticuloendothelial System><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><Cas nuclease technology><Cell Body><Cell Therapy><Cells><Chromosome Mapping><Clinical Treatment><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><Combining Site><DNA><DNA Alteration><DNA Sequence><DNA Sequence Alteration><DNA cassette><DNA mutation><Deoxyribonucleic Acid><Disease><Disorder><Dorsum><Endothelium><Engraftment><Extravasation><Face><Femur><Functional RNA><Future><Gene Alteration><Gene Delivery><Gene Localization><Gene Mapping><Gene Mapping Genetics><Gene Mutation><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Genetic mutation><Guide RNA><Hb SS disease><HbSS disease><Hematologic Diseases><Hematological Disease><Hematological Disorder><Hematopoiesis><Hematopoietic><Hematopoietic Cellular Control Mechanisms><Hematopoietic Progenitor Cells><Hematopoietic stem cells><Hemoglobin S Disease><Hemoglobin sickle cell disease><Hemoglobin sickle cell disorder><Hemoglobinopathies><Human><Immunochemical Immunologic><Immunologic><Immunological><Immunologically><Immunologics><In Situ><Infusion><Infusion procedures><Insect Viruses><Leakage><Linkage Mapping><Magnetic nanoparticles><Magnetism><Mammalian Cell><Mediating><Messenger RNA><Methods><Mice><Mice Mammals><Micro RNA><MicroRNAs><Modern Man><Murine><Mus><Mutation><Myelogenous><Myeloid><Nanotechnology><Non-Coding><Non-Coding RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Patients><Permeability><Population><Post-Translational Regulation><Posttranslational Regulation><Progenitor Cells><Reactive Site><Regulator Genes><Regulatory Element><Risk><Sequence Alteration><Sickle Cell Anemia><Site><Specificity><Spillage><System><Techniques><Therapeutic><Total Human and Non-Human Gene Mapping><Toxic effect><Toxicities><Transcriptional Regulatory Elements><Transfection><Translation Initiation><Transplantation><Untranslated RNA><Viral Vector><adaptive immunity><adulthood><autologous graft><autotransplantation><beta Globin><beta Thalassemia><blood cell formation><blood disorder><blood stem cell><cell mediated therapies><cell-based therapeutic><cell-based therapy><cellular therapeutic><cellular therapy><clinical applicability><clinical application><clinical translation><clinically translatable><complement system><cost><curative intervention><curative therapeutic><curative therapy><curative treatments><delivery vector><delivery vehicle><design><designing><enhancer cassette><expression cassette><faces><facial><gRNA><gene cassette><gene corrected><gene correction><genetic cassette><genetic mapping><genome mutation><genomic alteration><genotoxicity><hematopoietic progenitor><hematopoietic stem progenitor cell><hemopoietic><hemopoietic progenitor><hemopoietic stem cell><in vivo><infusions><integration cassette><intravenous injection><lipid based nanoparticle><lipid nanoparticle><mRNA><mRNA Translation><magnetic><magnetic field><miRNA><miRNAs><mouse model><multidisciplinary><murine model><myeloablation><nano medicinal><nano medicine><nano particle><nano tech><nano technology><nano-sized particle><nano-technological><nanomedicinal><nanomedicine><nanoparticle><nanosized particle><nanotech><nanotechnological><noncoding><novel><nuclease><p-Thalassemia><pre-clinical study><preclinical study><promoter cassette><regulatory gene><reporter cassette><resistance cassette><selectable cassette><selection cassette><self-renew><self-renewal><sickle cell disease><sickle cell disorder><sickle disease><sicklemia><site targeted delivery><stem cell biology><stem cells><stop cassette><success><synthetic biology><targeted delivery><therapeutic target><tool><trans acting element><transcription cassette><transcriptional cassette><transgene cassette><transgene delivery><translational opportunities><translational potential><transplant><trial regimen><trial treatment><vascular><vector><β-globin><β-thalassemia>