Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

High-Throughput Discovery and Characterization of Human Transcriptional Effectors.

Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.

Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma.

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

TRACE generates fluorescent human reporter cell lines to characterize epigenetic pathways.

Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.

CRISPR-Cas9 knockin mice for genome editing and cancer modeling.

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Neutral sphingomyelinase 2 is required for HIV-1 maturation.

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

Biologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease.

BACKGROUND: Sickle cell disease is characterized by the painful recurrence of vaso-occlusive events. Gene therapy with the use of LentiGlobin for sickle cell disease (bb1111; lovotibeglogene autotemcel) consists of autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified ß-globin gene, which produces an antisickling hemoglobin, HbAT87Q. METHODS: In this ongoing phase 1-2 study, we optimized the treatment process in the initial 7 patients in Group A and 2 patients in Group B with sickle cell disease. Group C was established for the pivotal evaluation of LentiGlobin for sickle cell disease, and we adopted a more stringent inclusion criterion that required a minimum of four severe vaso-occlusive events in the 24 months before enrollment. In this unprespecified interim analysis, we evaluated the safety and efficacy of LentiGlobin in 35 patients enrolled in Group C. Included in this analysis was the number of severe vaso-occlusive events after LentiGlobin infusion among patients with at least four vaso-occlusive events in the 24 months before enrollment and with at least 6 months of follow-up. RESULTS: As of February 2021, cell collection had been initiated in 43 patients in Group C; 35 received a LentiGlobin infusion, with a median follow-up of 17.3 months (range, 3.7 to 37.6). Engraftment occurred in all 35 patients. The median total hemoglobin level increased from 8.5 g per deciliter at baseline to 11 g or more per deciliter from 6 months through 36 months after infusion. HbAT87Q contributed at least 40% of total hemoglobin and was distributed across a mean (±SD) of 85±8% of red cells. Hemolysis markers were reduced. Among the 25 patients who could be evaluated, all had resolution of severe vaso-occlusive events, as compared with a median of 3.5 events per year (range, 2.0 to 13.5) in the 24 months before enrollment. Three patients had a nonserious adverse event related or possibly related to LentiGlobin that resolved within 1 week after onset. No cases of hematologic cancer were observed during up to 37.6 months of follow-up. CONCLUSIONS: One-time treatment with LentiGlobin resulted in sustained production of HbAT87Q in most red cells, leading to reduced hemolysis and complete resolution of severe vaso-occlusive events. (Funded by Bluebird Bio; HGB-206 ClinicalTrials.gov number, NCT02140554.).

Acute Myeloid Leukemia Case after Gene Therapy for Sickle Cell Disease.

Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the ßA-T87Q-globin gene. Acute myeloid leukemia developed in a woman approximately 5.5 years after she had received LentiGlobin for sickle cell disease as part of the initial cohort (Group A) of the HGB-206 study. An analysis of peripheral-blood samples revealed that blast cells contained a BB305 lentiviral vector insertion site. The results of an investigation of causality indicated that the leukemia was unlikely to be related to vector insertion, given the location of the insertion site, the very low transgene expression in blast cells, and the lack of an effect on expression of surrounding genes. Several somatic mutations predisposing to acute myeloid leukemia were present after diagnosis, which suggests that patients with sickle cell disease are at increased risk for hematologic malignant conditions after transplantation, most likely because of a combination of risks associated with underlying sickle cell disease, transplantation procedure, and inadequate disease control after treatment. (Funded by Bluebird Bio.).

Betibeglogene Autotemcel Gene Therapy for Non-ß0/ß0 Genotype ß-Thalassemia.

BACKGROUND: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent ß-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the ß-globin (ßA-T87Q) gene. METHODS: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent ß-thalassemia and a non-ß0/ß0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months). RESULTS: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed. CONCLUSIONS: Treatment with beti-cel resulted in a sustained HbAT87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-ß0/ß0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.).

Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

Antigen identification and high-throughput interaction mapping by reprogramming viral entry.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

Capsid-dependent lentiviral restrictions.

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5, have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

The AIM2-like Receptors Are Dispensable for the Interferon Response to Intracellular DNA.

Detection of intracellular DNA triggers activation of the STING-dependent interferon-stimulatory DNA (ISD) pathway, which is essential for antiviral responses. Multiple DNA sensors have been proposed to activate this pathway, including AIM2-like receptors (ALRs). Whether the ALRs are essential for activation of this pathway remains unknown. To rigorously explore the function of ALRs, we generated mice lacking all 13 ALR genes. We found that ALRs are dispensable for the type I interferon (IFN) response to transfected DNA ligands, DNA virus infection, and lentivirus infection. We also found that ALRs do not contribute to autoimmune disease in the Trex1(-/-) mouse model of Aicardi-Goutières Syndrome. Finally, CRISPR-mediated disruption of the human AIM2-like receptor IFI16 in primary fibroblasts revealed that IFI16 is not essential for the IFN response to human cytomegalovirus infection. Our findings indicate that ALRs are dispensable for the ISD response and suggest that alternative functions for these receptors should be explored.

Tetherin: holding on and letting go.

Mammalian cells are equipped with so-called "restriction factors" that suppress virus replication and help to prevent virus transmission from one species to another. This Essay discusses the host restriction factor tetherin, which blocks the release of enveloped viruses like HIV-1, and the factors evolved by primate lentiviruses, such as Vpu and Nef, that antagonize tetherin's action.

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells.

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.