Optimizing CRISPR/Cas9 genome editing in primary human hematopoietic cells to advance studies into HIV biology

Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.

A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.

CD4-directed nanoblades enable selective genome editing in CD4+ cells and HIV suppression in vitro and in vivo

Current antiretroviral therapies suppress HIV replication but fail to eliminate integrated proviral DNA in long-lived CD4⁺ cells, precluding a cure. CRISPR-Cas9 offers potential for HIV eradication but efficient and cell-specific delivery into HIV target cells remains a major hurdle. We developed CD4-directed Nanoblades (CD4-NBs), murine leukemia virus-like particles pseudotyped with anti-CD4 nanobodies and a fusogenic glycoprotein VSV Gmut, to selectively deliver Cas9-gRNA ribonucleoproteins into CD4⁺ cells. CD4-NBs selectively delivered cargo to CD4⁺ cells in vitro and in vivo, achieving efficient gene disruption in primary CD4+ cells. Dual-guide CD4-NBs targeting conserved HIV tat/rev/env regions disrupted proviral DNA, suppressing HIV infection in CD4+ cells. In HIV-infected, ART-pretreated humanized mice, CD4-NBs significantly reduced plasma viremia. While full tissue reservoir clearance was not achieved, repeated dosing did reduce viral RNA and proviral DNA in bone marrow and lungs, respectively. As such, this proof-of-concept study supports the promise of CD4-NBs as a minimally invasive, CD4⁺ cell-targeted gene editing strategy for HIV therapy. Virus-like particles engineered with anti-CD4 nanobodies (i.e., CD4-directed nanoblades) present a promising HIV cure strategy as they achieve CD4+ cell-specific CRISPR-Cas9 delivery, efficient gene editing, HIV proviral DNA disruption and reduced viremia in ART-pretreated humanized mice. CD4-NBs were engineered by pseudotyping murine leukemia virus-like particles with anti-CD4 nanobodies and a fusogenic glycoprotein to enable targeted Cas9-gRNA ribonucleoprotein delivery. Selective cargo delivery was achieved in vitro and in vivo, with efficient gene disruption observed specifically in (primary) CD4⁺ cells. HIV proviral DNA was disrupted using dual-guide CD4-NBs targeting conserved HIV tat/rev/env regions, resulting in suppressed HIV infection in CD4⁺ cells. In HIV-infected, ART-pretreated humanized mice, viremia was significantly reduced following CD4-NB administration. Repeated CD4-NB dosing was associated with reductions in viral RNA and proviral DNA in bone marrow and lungs, respectively, though full tissue reservoir clearance was not achieved. CD4-NBs were engineered by pseudotyping murine leukemia virus-like particles with anti-CD4 nanobodies and a fusogenic glycoprotein to enable targeted Cas9-gRNA ribonucleoprotein delivery. Selective cargo delivery was achieved in vitro and in vivo, with efficient gene disruption observed specifically in (primary) CD4⁺ cells. HIV proviral DNA was disrupted using dual-guide CD4-NBs targeting conserved HIV tat/rev/env regions, resulting in suppressed HIV infection in CD4⁺ cells. In HIV-infected, ART-pretreated humanized mice, viremia was significantly reduced following CD4-NB administration. Repeated CD4-NB dosing was associated with reductions in viral RNA and proviral DNA in bone marrow and lungs, respectively, though full tissue reservoir clearance was not achieved. Virus-like particles engineered with anti-CD4 nanobodies (i.e., CD4-directed nanoblades) present a promising HIV cure strategy as they achieve CD4+ cell-specific CRISPR-Cas9 delivery, efficient gene editing, HIV proviral DNA disruption and reduced viremia in ART-pretreated humanized mice.

A CRISPR-Cas9 platform for primary human hepatocytes enables arrayed screening and in vivo validation of HBV host factors

Lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH) are reported, a system that enables PHH expansion in liver-humanized mice and enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.

CRISPR/Cas9-based repair of a heterozygous HNF1A mutation in patient-derived hiPSCs

Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.

Efficient CRISPR/Cas9-mediated homology independent sequence replacement in vivo and non-dividing primary cells

Base Editing Hematopoietic Stem/Progenitor Cell Gene Therapy for Treatment of X-Linked Severe Combined Immunodeficiency

X-linked Severe Combined Immunodeficiency (XSCID) due to IL2RG loss-of-function mutations typically cause a severe combined cellular and humoral immunodeficiency that is diagnosed and transplanted in infancy. Hypomorphic IL2RG variants with milder phenotypes are generally diagnosed later in life. Early stem cell transplants (allogeneic or autologous gene therapy) performed without conditioning often results in partial immune reconstitution with limited donor engraftment in T cell lineage alone. Defective host B, natural killer (NK), and other cell lineages remain and can cause progressive multi-organ disease. Ex vivo gene therapy using lentivector-transduced autologous hematopoietic stem/progenitor cells (HSPCs) has provided clinical benefit for many previously transplanted older XSCID patients (NCT01306019) and transplant-naïve infants (NCT01512888, NCT03311503). Lentivector transduction functionally corrects XSCID stem cells by semi-random insertion of IL2RG cDNA under the regulation of an engineered promoter. A conversion to gene-corrected immune cells in their respective compartment can take years, particularly in older adult patients. The exact mechanism for this slow progression, whether this is attributable to a relatively weak promoter in the transgenes, is unclear. CRISPR/Cas9 base editing provides the potential for precise gene mutation correction that is devoid of random virus integrations and, critically, restores physiological gene regulation by endogenous promoter. We developed a highly efficient base editing strategy using adenine base editor and respective guide RNAs to repair respective IL2RG mutations (IL2RG p.289X, IL2RG p.Gln235X, IL2RG p.Q144X, and IL2RG p.R226H). Preclinical efficacy and safety data supported a Phase I/II clinical trial to treat XSCID patients with base-edited HSPCs (IND 31037; NCT 06851767). Three patients have been treated to date with base-edited (BE) HSPCs (25 million to 50 million cells per kg/weight). BE HSPC products were well tolerated, with early myeloid recovery within 3 weeks after cell infusion. Gene correction frequencies in the study cell products averaged >80%. Follow-up data at 6 months in the first patient showed robust levels of gene correction in myeloid (70%), B (94%), NK (100%), and, surprisingly, in his T cell compartment (65%) as well, with a corresponding decline in donor T cell chimerism (99% at baseline, 47% at 6 months). Monitoring of the other treated patients is ongoing. We conclude from our preliminary data that gene therapy using BE HSPCs appear very promising for achieving an earlier and more robust multi-lineage immune reconstitution in adult patients using BE HSPCs.