Biology subjects

Ghanim, H. Y.

Publications and source records attributed to Ghanim, H. Y..

4 recordsLinked to original sources

bioRxiv2025

Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

Canonical HIV-1 entry into target cells depends on binding to CD4 as a primary receptor. Because of this, use of the CD4 receptor as a viral trap (a decoy receptor used to prevent infection of target cells) is a promising strategy for the treatment of HIV-1. One challenge in using CD4 viral traps is maintaining enough of the decoy receptor in circulation to remain effective. Here we present a strategy to produce cell-based CD4 viral traps by engineering hematopoietic stem and progenitor cells (HSPCs) to express the CD4 receptor in red blood cell (RBC) progeny. This takes advantage of the ability of the HSPC to repopulate the blood system for a lifetime, while leveraging the fact that RBCs greatly outnumber any cell targeted for infection. Engineered HSPCs efficiently express CD4 on their cell surface after differentiation to the RBC lineage in vitro. Fusion of CD4 to glycophorin A (GPA) and introduction of a truncated erythropoietin receptor (tEPOR) leads to increased CD4 expression and enrichment of edited cells (CD4-GPA-tEPOR) to levels capable of neutralizing HIV-1 pseudovirus in vitro. In sum, this work presents a potential strategy for the one-time delivery of CD4-RBC viral traps through autologous transplantation of engineered HSPCs.

bioengineering↗

bioRxiv2024

Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies

Autologous transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously. Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

bioengineering↗

bioRxiv2022

Genetically Corrected RAG2-SCID Human Hematopoietic Stem CellsRestore V(D)J-Recombinase and Rescue Lymphoid Deficiency

Recombination-activating genes (RAG1 and RAG2) are critical in lymphoid cell development and function for initiating the V(D)J-recombination process to generate polyclonal lymphocytes with broad antigen-specificity. Clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCID), causing life-threatening infections and death early in life in the absence of hematopoietic cell transplantation (HCT). Haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phases of the cell cycle at the early stages of T and B cell development, underscoring that a direct gene correction would capture the precise temporal expression of the endogenous gene, is a promising therapeutic approach for RAG1/2-deficiencies. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) in healthy donors and one RAG2-SCID patient. V(D)J recombinase activity was restored following gene correction of RAG2-SCID-derived HSPCs, resulting in the development of TCR {beta} and {gamma}{delta} CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of the CDR3 length and preserved usage of distal TRAV genes. We confirmed in vivo rescue of B-cell development, with normal IgM surface expression and a significant decrease in CD56bright NK cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit all RAG2-deficient patients. KEY POINTSO_LIHuman hematopoietic stem cells can be corrected to restore endogenous RAG2 gene expression while preserving durable engraftment potential. C_LIO_LIGene-corrected RAG2 locus restores V(D)J recombination in RAG2-SCID patient stem cells, promoting T and B-cells receptor formation. C_LI

immunology↗

bioRxiv2021

Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

genetics↗