Obesity-associated gene TMEM18 has a role in the central control of appetite and body weight regulation.

An intergenic region of human chromosome 2 (2p25.3) harbors genetic variants which are among those most strongly and reproducibly associated with obesity. The gene closest to these variants is TMEM18, although the molecular mechanisms mediating these effects remain entirely unknown. Tmem18 expression in the murine hypothalamic paraventricular nucleus (PVN) was altered by changes in nutritional state. Germline loss of Tmem18 in mice resulted in increased body weight, which was exacerbated by high fat diet and driven by increased food intake. Selective overexpression of Tmem18 in the PVN of wild-type mice reduced food intake and also increased energy expenditure. We provide evidence that TMEM18 has four, not three, transmembrane domains and that it physically interacts with key components of the nuclear pore complex. Our data support the hypothesis that TMEM18 itself, acting within the central nervous system, is a plausible mediator of the impact of adjacent genetic variation on human adiposity.

Stability of the adeno-associated virus 8 reference standard material.

Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions ( <-70° C). The use of RSM is strongly recommended and endorsed by regulatory agencies to normalize laboratory internal controls and to provide accurate titration of AAV vectors lots.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Liver production of sulfamidase reverses peripheral and ameliorates CNS pathology in mucopolysaccharidosis IIIA mice.

Mucopolysaccharidosis type IIIA (MPSIIIA) is an inherited lysosomal storage disease caused by deficiency of sulfamidase, resulting in accumulation of the glycosaminoglycan (GAG) heparan sulfate. It is characterized by severe progressive neurodegeneration, together with somatic alterations, which lead to death during adolescence. Here, we tested the ability of adeno-associated virus (AAV) vector-mediated genetic modification of either skeletal muscle or liver to revert the already established disease phenotype of 2-month-old MPSIIIA males and females. Intramuscular administration of AAV-Sulfamidase failed to achieve significant therapeutic benefit in either gender. In contrast, AAV8-mediated liver-directed gene transfer achieved high and sustained levels of circulating active sulfamidase, which reached normal levels in females and was fourfold higher in males, and completely corrected lysosomal GAG accumulation in most somatic tissues. Remarkably, a 50% reduction of GAG accumulation was achieved throughout the entire brain of males, which correlated with a partial improvement of the pathology of cerebellum and cortex. Liver-directed gene transfer expanded the lifespan of MPSIIIA males, underscoring the importance of reaching supraphysiological plasma levels of enzyme for maximal therapeutic benefit. These results show how liver-directed gene transfer can reverse somatic and ameliorate neurological pathology in MPSIIIA.

Human and Insect Cell-Produced Recombinant Adeno-Associated Viruses Show Differences in Genome Heterogeneity.

In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing "inaccurate" vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in Spodoptera frugiperda insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these "mutated" ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates.

Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application.

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers.

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.

Homologous Recombination Offers Advantages over Transposition-Based Systems to Generate Recombinant Baculovirus for Adeno-Associated Viral Vector Production.

Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints. Moreover, baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raises concerns about safety due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, a faster and safer method based on homologous recombination (HR) is investigated. First, the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes are confirmed. Strikingly, it is found that the exogenous cassette showed increased stability over passages when using the HR system. Finally, both materials generated high rAAV vector genome titers, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for its manufacturing. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products.

Baboon Envelope Pseudotyped "Nanoblades" Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells.

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized "nanoblades," a new technology that delivers a genomic cleaving agent into cells. These are modified murine leukemia virus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structural protein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs. Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells. Here, we showed that nanoblades were remarkably efficient for entry into human T, B, and hematopoietic stem and progenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboon retroviral and VSV-G envelope glycoproteins. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34+ cells (HSPCs) treated with the same nanoblades allowed 30-40% exon 1 drop-out in the WAS gene locus. Importantly, no toxicity was detected upon nanoblade-mediated gene editing of these blood cells. Finally, we also treated HSPCs with nanoblades in combination with a donor-encoding rAAV6 vector resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. Summarizing, this new technology is simple to implement, shows high flexibility for different targets including primary immune cells of human and murine origin, is relatively inexpensive and therefore gives important prospects for basic and clinical translation in the area of gene therapy.

Increased intraocular insulin-like growth factor-I triggers blood-retinal barrier breakdown.

Blood-retinal barrier (BRB) breakdown is a key event in diabetic retinopathy and other ocular disorders that leads to increased retinal vascular permeability. This causes edema and tissue damage resulting in visual impairment. Insulin-like growth factor-I (IGF-I) is involved in these processes, although the relative contribution of increased systemic versus intraocular IGF-I remains controversial. Here, to elucidate the role of this factor in BRB breakdown, transgenic mice with either local or systemic elevations of IGF-I have been examined. High intraocular IGF-I, resulting from overexpression of IGF-I in the retina, increased IGF-I receptor content and signaling and led to accumulation of vascular endothelial growth factor. This was parallel to up-regulation of vascular Intercellular adhesion molecule I and retinal infiltration by bone marrow-derived microglial cells. These alterations resulted in increased vessel paracellular permeability to both low and high molecular weight compounds in IGF-I-overexpressing retinas and agreed with the loss of vascular tight junction integrity observed by electron microscopy and the altered junctional protein content. In contrast, mice with chronically elevated serum IGF-I did not show alterations in the retinal vasculature structure and permeability, indicating that circulating IGF-I cannot initiate BRB breakdown. Consistent with a key role of IGF-I signaling in retinal diseases, a strong up-regulation of the IGF-I receptor in human retinas with marked gliosis was also observed. Thus, this study demonstrates that intraocular IGF-I, but not systemic IGF-I, is sufficient to trigger processes leading to BRB breakdown and increased retinal vascular permeability. Therefore, therapeutic interventions designed to counteract local IGF-I effects may prove successful to prevent BRB disruption.

Endothelial cell transduction in primary cultures from regressing mesonephros.

Loss of renal function during normal aging is associated with vascular alterations. Consequently, new therapeutic approaches, including gene therapy, to protect renal endothelial cells are expected to be greatly beneficial. Quail mesonephros is a transitory embryonic kidney that has been used for the study of vascular development and involution. Vascular alterations in regressing mesonephros are similar to those observed in aging kidney. In the present study, we examined adenovirus-mediated gene transfer to endothelial cells in primary cultures from developing and regressing quail mesonephros. Quail embryos with developing and regressing mesonephros were examined on day 6 (30HH) and day 11 (40HH) of incubation, respectively. The senescence markers, associated beta-galactosidase activity and p16(INK4a), were examined in whole mesonephros. Quail embryos were injected intracardiacally with adenoviral vectors (rAd-CMV-LacZ) and endothelial cell transduction examined. In addition, primary cell cultures from mesonephros were exposed to adenoviral vectors. Endothelial cells in primary cultures were identified as QH1(+), LEP100(-) and acidic phosphatase(-) cells and adenovirus-transduced cells were those positive for bacterial-associated beta-galactosidase activity. We report that endothelial cells in the whole regressing mesonephros and primary cell cultures expressed senescence markers. In addition, we observed that adenoviral vectors were able to transduce endothelial cells in the whole regressing mesonephros, and that cultured endothelial and macrophagic cells from the regressing mesonephros were more efficiently transduced than those derived from the developing mesonephros. Our results suggest that quail mesonephros provides a practical model to assay gene transfer to endothelial cells in regressing/senescent vessels.

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes.

Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.

Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.

Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes.

With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

Chemical modification of the adeno-associated virus capsid to improve gene delivery.

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells and sustained maintenance of the viral genome. However, the conclusion from clinical data using these vectors is that there is a need to develop new AAVs with a higher transduction efficiency and specificity for relevant target tissues. To overcome these limitations, we chemically modified the surface of the capsid of AAV vectors. These modifications were achieved by chemical coupling of a ligand by the formation of a thiourea functionality between the amino group of the capsid proteins and the reactive isothiocyanate motif incorporated into the ligand. This strategy does not require genetic engineering of the capsid sequence. The proof of concept was first evidenced using a fluorophore (FITC). Next, we coupled the N-acetylgalactosamine ligand onto the surface of the AAV capsid for asialoglycoprotein receptor-mediated hepatocyte-targeted delivery. Chemically-modified capsids also showed reduced interactions with neutralizing antibodies. Taken together, our findings reveal the possibility of creating a specific engineered platform for targeting AAVs via chemical coupling.

Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.

Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.