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

Orthogonal CRISPR screens and human liver chimeric mice identify hepatitis B virus host factors

Hepatitis B virus (HBV) chronically infects approximately 250 million people worldwide, and reliable curative therapies are lacking. A broader understanding of viral-host interactions could accelerate efforts to find new host-centric therapeutic targets. However, inefficient cell culture systems and limited replication markers compatible with pooled screening have precluded the widespread use of genetic perturbation screens. Here, we performed the first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection and integrated these results with two orthogonal pooled screens to identify host factors. We selected 72 genes for a multi-step assessment that included arrayed validation assays using both HBV infection and pgRNA transfection. We then independently tested thirteen genes using high-efficiency bulk KO experiments to guide further investigations of both antiviral and proviral factors. In both KO and siRNA-mediated knockdown experiments, depletion of the top antiviral factor, EXOC1, enhanced multiple HBV replication markers, and transcriptomic analysis revealed activation of hypoxia- and HIF-1 gene signatures. Three proviral factors, IRF2, WDR48, and ZCCHC14, were investigated in vivo using a human liver chimeric mouse model, which demonstrated that ZCCHC14 KO greatly reduced HBV replication and spread. Together, these complementary in vitro and in vivo platforms expand the catalog of HBV host factors and provide a scalable framework for host target discovery.

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.

Identification of AAV Vector Integration Sites in Primary Human and Macaque Hepatocytes in vivo, and Analysis of Oncogenic Risk.

Recombinant adeno-associated virus (rAAV) vectors are widely used for in vivo gene therapy, yet their potential to integrate into the host genome raises concerns about insertional mutagenesis and oncogenic risk, particularly in the liver where vector exposure is highest. To address this, we analyzed rAAV integration patterns in primary human hepatocytes xenografted into FRG mouse livers and in hepatocytes from cynomolgus macaques following systemic rAAV administration. High-resolution integration site mapping yielded approximately 1.5 million and 1.3 million unambiguously mapped sites in human and macaque genomes, respectively. Both datasets revealed a bias toward integration within transcriptionally active genes and regions of open chromatin, consistent with previous reports, but no particular preference for genes implicated in hepatocellular carcinoma (HCC) was observed. While numerous common integration sites (CIS) were identified, their distribution differed between species. Notably, a CIS was observed at the AAVS1 locus in human hepatocytes, raising the possibility of Rep-mediated integration. These findings highlight the need for continued monitoring of integration events in clinical settings. Overall, the data support a low oncogenic risk profile for the evaluated vector while reinforcing the value of direct human liver integration analyses to refine risk assessment and guide the development of safer gene therapy platforms.

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.

Functional evaluation of a natural AAV capsid liver targeting motif in human hepatocytes

Background&Aims Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods Two human hepatocyte models were tested: Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.