The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

Liposome-lentivirus for miRNA therapy with molecular mechanism study.

BACKGROUND: Cancer stem cells (CSCs) play a vital role in the occurrence, maintenance, and recurrence of solid tumors. Although, miR-145-5p can inhibit CSCs survival, poor understanding of the underlying mechanisms hamperes further therapeutic optimization for patients. Lentivirus with remarkable transduction efficiency is the most commonly used RNA carrier in research, but has shown limited tumor-targeting capability. METHODS: We have applied liposome to decorate lentivirus surface thereby yielding liposome-lentivirus hybrid-based carriers, termed miR-145-5p-lentivirus nanoliposome (MRL145), and systematically analyzed their potential therapeutic effects on liver CSCs (LCSCs). RESULTS: MRL145 exhibited high delivery efficiency and potent anti-tumor efficacy under in vitro and in vivo. Mechanistically, the overexpressed miR-145-5p can significantly suppress the self-renewal, migration, and invasion abilities of LCSCs by targeting Collagen Type IV Alpha 3 Chain (COL4A3). Importantly, COL4A3 can promote phosphorylating GSK-3ß at ser 9 (p-GSK-3ß S9) to inactivate GSK3ß, and facilitate translocation of ß-catenin into the nucleus to activate the Wnt/ß-catenin pathway, thereby promoting self-renewal, migration, and invasion of LCSCs. Interestingly, COL4A3 could attenuate the cellular autophagy through modulating GSK3ß/Gli3/VMP1 axis to promote self-renewal, migration, and invasion of LCSCs. CONCLUSIONS: These findings provide new insights in mode of action of miR-145-5p in LCSCs therapy and indicates that liposome-virus hybrid carriers hold great promise in miRNA delivery.

Towards a Cure for Diamond-Blackfan Anemia: Views on Gene Therapy.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.

Prospects and limitations of cumate-inducible lentivirus as a tool for investigating VEGF-A-mediated pathology in diabetic retinopathy.

Diabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR. In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability. Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry. Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis. In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mechanistic studies in cellular drug discovery, its use is not a feasible approach to model DR in in vivo studies due to cumate-induced retinal toxicity.

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Non-clinical safety assessment of novel drug modalities: Genome safety perspectives on viral-, nuclease- and nucleotide-based gene therapies.

Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.

Surface-modified injectable poly(ethylene-glycol) diacrylate-based cryogels for localized gene delivery.

Lentiviral transduction is widely used in research, has shown promise in clinical trials involving gene therapy and has been approved for CAR-T cell immunotherapy. However, most modifications are doneex vivoand rely on systemic administration of large numbers of transduced cells for clinical applications. A novel approach utilizingin situbiomaterial-based gene delivery can reduce off-target side effects while enhancing effectiveness of the manipulation process. In this study, poly(ethylene glycol) diacrylate (PEGDA)-based scaffolds were developed to enablein situlentivirus-mediated transduction. Compared to other widely popular biomaterials, PEGDA stands out due to its robustness and cost-effectiveness. These scaffolds, prepared via cryogelation, are capable of flowing through surgical needles in bothin vitroandin vivoconditions, and promptly regain their original shape. Modification with poly(L-lysine) (PLL) enables lentivirus immobilization while interconnected macroporous structure allows cell infiltration into these matrices, thereby facilitating cell-virus interaction over a large surface area for efficient transduction. Notably, these preformed injectable scaffolds demonstrate hemocompatibility, cell viability and minimally inflammatory response as shown by ourin vitroandin vivostudies involving histology and immunophenotyping of infiltrating cells. This study marks the first instance of using preformed injectable scaffolds for delivery of lentivectors, which offers a non-invasive and localized approach for delivery of factors enablingin situlentiviral transduction suitable for both tissue engineering and immunotherapeutic applications.

[In vitro and in vivo validation of cytotoxicity of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells].

Objective To evaluate the toxicology of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells and to provide a safety basis for the clinical evaluation of HER2-CAR-T cell therapy. Methods The recombinant lentiviral vector was used to generate HER2-CAR-T cells. Soft agar colony formation assay was used to observe the colony formation of HER2-CAR-T cells, and the colony formation rate was statistically analyzed. The HER2-CAR-T cell suspension was co-incubated with rabbit red blood cell suspension, and the hemolysis of red blood cells was evaluated by direct observation and microplate reader detection. The HER2-CAR-T cell preparation was injected into the ear vein of male New Zealand rabbits, and the stimulating effect of HER2-CAR-T cells on the blood vessels of the animals was observed by staining of tissue sections. The vesicular stomatitis virus envelope glycoprotein (VSV-G) gene of pMD 2.G vector was used as the target sequence, and the safety of the lentiviral vector was verified by real-time fluorescence quantitative PCR. The heart, liver, lung, and kidney of mice receiving HER2-CAR-T cell infusion were collected, and the lesions were observed by HE staining. Results The HER2-CAR-T cells were successfully prepared. These cells did not exhibit soft agar colony formation ability in vitro, and the HER2-CAR-T cell preparation did not cause hemolysis in New Zealand rabbit red blood cells. After the infusion of HER2-CAR-T cells into the ear vein of New Zealand rabbits, no obvious vascular stimulation response was found, and no specific amplification of VSV-G was detected. No obvious lesions were found in the heart, liver, lung and kidney tissues of the treatment group. Conclusion The prepared HER2-CAR-T cells have reliable safety.

Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia.

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Checkpoint inhibitor-expressing lentiviral vaccine suppresses tumor growth in preclinical cancer models.

BACKGROUND: While immunotherapy has been highly successful for the treatment of some cancers, for others, the immune response to tumor antigens is weak leading to treatment failure. The resistance of tumors to checkpoint inhibitor therapy may be caused by T cell exhaustion resulting from checkpoint activation. METHODS: In this study, lentiviral vectors that expressed T cell epitopes of an experimentally introduced tumor antigen, ovalbumin, or the endogenous tumor antigen, Trp1 were developed. The vectors coexpressed CD40 ligand (CD40L), which served to mature the dendritic cells (DCs), and a soluble programmed cell death protein 1 (PD-1) microbody to prevent checkpoint activation. Vaccination of mice bearing B16.OVA melanomas with vector-transduced DCs induced the proliferation and activation of functional, antigen-specific, cytolytic CD8 T cells. RESULTS: Vaccination induced the expansion of CD8 T cells that infiltrated the tumors to suppress tumor growth. Vector-encoded CD40L and PD-1 microbody increased the extent of tumor growth suppression. Adoptive transfer demonstrated that the effect was mediated by CD8 T cells. Direct injection of the vector, without the need for ex vivo transduction of DCs, was also effective. CONCLUSIONS: This study suggests that therapeutic vaccination that induces tumor antigen-specific CD8 T cells coupled with a vector-expressed checkpoint inhibitor can be an effective means to suppress the growth of tumors that are resistant to conventional immunotherapy.

Lentivirus-Mediated BCL-XL Overexpression Inhibits Stem Cell Apoptosis during Ex Vivo Expansion and Provides Competitive Advantage Following Xenotransplantation.

Hematopoietic reconstitution after hematopoietic stem cell transplantation (HSCT) is influenced by the number of transplanted cells. However, under certain conditions donor cell counts are limited and impair clinical outcome. Hematopoietic stem and progenitor cell (HSPC) expansion prior to HSCT is a widely used method to achieve higher donor cell counts and minimize transplantation-related risks such as graft failure or delayed engraftment. Still, expansion in a non-physiological environment can trigger cell death mechanisms and hence counteract the desired effect. We have shown earlier that during HSCT a relevant amount of HSPCs were lost due to apoptosis and that cell death inhibition in donor HSPCs improved engraftment in xenotransplantation experiments. Here, we assessed the effect of combined ex vivo expansion and cell death inhibition on HSPC yield and their reconstitution potential in vivo. During expansion with cytokines and the small molecule inhibitor StemRegenin 1, concomitant lentiviral overexpression of antiapoptotic BCL-XL resulted in an increased yield of transduced HSPCs. Importantly, BCL-XL overexpression enhanced the reconstitution potential of HSPCs in xenotransplantation experiments in vivo. In contrast, treatment with caspase and necroptosis inhibitors had no favorable effects on HSPC yields nor on cell viability. We postulate that overexpression of antiapoptotic BCL-XL, both during ex vivo expansion and transplantation, is a promising approach to improve the outcome of HSCT in situations with limited donor cell numbers. However, such apoptosis inhibition needs to be transient to avoid long-term sequelae like leukemia.

GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice.

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells.

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional ß-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). METHODS: After HSPCs were transduced with an LV encoding the therapeutic ß-globin (ßA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. RESULTS: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of ßA-T87Q protein detected by reverse-phase high-performance liquid chromatography. CONCLUSIONS: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

OBJECTIVES: We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-ß3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. RESULTS: The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-ß3 more efficiently and optimizing the appropriate parameters, including concentration and duration. CONCLUSIONS: The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-ß3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

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.

Envelope protein-specific B cell receptors direct lentiviral vector tropism in vivo.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

High yield sterile filtration process for highly concentrated lentiviral vectors.

The development and manufacture of biopharmaceuticals are subject to strict regulations that specify the required minimum quality of the products. A key measure to meet these quality requirements is the integration of a sterile filtration step into the commercial manufacturing process. Whereas common procedures for most biologics exist, this is challenging for lentiviral vector (LVV) production for ex vivo gene therapy. LVVs nominal size is more than half the pore size (0.2 µm) of filters used for sterile filtration. Hence, highly concentrated virus solutions are prone to filter clogging if aggregation of viruses occurs or impurities attach to the viruses. Several filters were screened aiming to identify those which allow filtering highly concentrated stocks of LVVs of up to 1E + 9 transducing units mL-1 , which corresponds to 4.5E + 12 particles mL-1 . In addition, the effect of endonuclease treatment upstream of the purification process on filter performance was studied. In summary, three suitable filters were identified in a small-scale study (<15 mL) with virus yields >80% and the process was successfully scaled-up to a final scale of 100 mL LVV stock solution.

Bioinformatics Insights on Viral Gene Expression Transactivation: From HIV-1 to SARS-CoV-2.

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.