Jonas Holst Wolff

GATA2 (GATA binding protein 2) deficiency is a severe immunodeficiency caused by heterozygous variants in the gene encoding the transcription factor GATA2. Ex vivo gene editing of a patient’s own CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs) could provide curative treatment. However, current methods that rely on nuclease-dependent editing face considerable challenges, including off-target effects, genotoxicity, and reduced engraftment potential. Here, we report the development of an efficient gene editing therapy for GATA2 deficiency using prime editing with a favorable safety profile in terms of off-target effects and genotoxicity. We used prime editing to correct a GATA2 c.956_962del variant in patient-derived CD34 ⁺ HSPCs, reaching up to 70% prime editing efficiency and an increase in functional GATA2 alleles from 50 to 77%. We demonstrate that prime-edited patient HSPCs showed increased engraftment potential compared with untreated cells and detected limited on-target genotoxicity and no off-target editing at the top 20 predicted sites. Short prestimulation of CD34 ⁺ HSPCs supported efficient prime editing while preserving stemness, mitigating p53 activation, and increasing multilineage engraftment. We report PASSIGE (prime editing–assisted site-specific integrase gene editing) in CD34 ⁺ HSPCs, developing a more broadly applicable, double-strand break–independent complementary DNA insertion strategy with the potential to address a large proportion of alleles causing GATA2 deficiency. Together, our results demonstrate the preclinical development of prime editing–based therapies for GATA2 deficiency in CD34 ⁺ HSPCs.

Inborn errors of immunity (IEI) impairing brain-intrinsic immune defenses can underlie herpes simplex virus encephalitis. By whole-exome sequencing of cohorts of herpesvirus-associated recurrent lymphocytic meningitis and acute retinal necrosis, we identified two patients heterozygous for variants in interferon (IFN) regulatory factor 7 (IRF7). The expression of the Q185X (patient 1, P1) and A86Rfs23X (P2) IRF7 variants in HEK293T cells resulted in truncated IRF7 proteins that lacked IFN-transactivating ability. Peripheral blood mononuclear cells from P1 exhibited reduced type I IFN responses to HSV-2 infection. Genetic knock-in of the IRF7 Q185X variant in THP-1 cells and stem cell–derived plasmacytoid dendritic cells (pDC) confirmed the disrupted IFN expression, resulting in impaired paracrine antiviral protection of meningeal fibroblasts. Strikingly, genetically heterozygous index patient pDC, but not those of healthy carrier family members, showed expression of only the pathogenic IRF7 Q185X allele, resulting in a homozygous transcriptotype. Collectively, this study identifies genetically heterozygous but transcriptionally homozygous IRF7 deficiency as an IEI underlying herpesvirus central nervous system infection.

Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34⁺ hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.

Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such ‘all-in-one’ LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

Viruses are naturally endowed with the capacity to transfer genetic material between cells. Following early skepticism, engineered viruses have been used to transfer genetic information into thousands of patients, and genetic therapies are currently attracting large investments. Despite challenges and severe adverse effects along the way, optimized technologies and improved manufacturing processes are driving gene therapy toward clinical translation. Fueled by the outbreak of AIDS in the 1980s and the accompanying focus on human immunodeficiency virus (HIV), lentiviral vectors derived from HIV have grown to become one of the most successful and widely used vector technologies. In 2022, this vector technology has been around for more than 25 years. Here, we celebrate the anniversary by portraying the vector system and its intriguing properties. We dive into the technology itself and recapitulate the use of lentiviral vectors for ex vivo gene transfer to hematopoietic stem cells and for production of CAR T-cells. Furthermore, we describe the adaptation of lentiviral vectors for in vivo gene delivery and cover the important contribution of lentiviral vectors to basic molecular research including their role as carriers of CRISPR genome editing technologies. Last, we dwell on the emerging capacity of lentiviral particles to package and transfer foreign proteins.