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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.

[Display omitted] This was a single-arm, multicenter, open-label phase I trial. Lentiviral vectors (LV) carrying the ABCD1 gene (LV- ABCD1) was directly injected into the brain of patients with childhood cerebral adrenoleukodystrophy (CCALD), and multi-site injection was performed. The injection dose increased from 200 to 1600 μL (vector titer: 1×109 transduction units per mL (TU/mL)), and the average dose per kilogram body weight ranges from 8 to 63.6 μL/kg. The primary endpoint was safety, dose‐exploration and immunogenicity and the secondary endpoint was initial evaluation of efficacy and the expression of ABCD1 protein. A total of 7 patients participated in this phase I study and were followed for 1 year. No injection-related serious adverse event or death occurred. Common adverse events associated with the injection were irritability (71%, 5/7) and fever (37.2–38.5 ℃, 57%, 4/7). Adverse events were mild and self-limited, or resolved within 3 d of symptomatic treatment. The maximal tolerable dose is 1600 μL. In 5 cases (83.3%, 5/6), no lentivirus associated antibodies were detected. The overall survival at 1-year was 100%. The ABCD1 protein expression was detected in neutrophils, monocytes and lymphocytes. This study suggests that the intracerebral injection of LV- ABCD1 for CCALD is safe and can achieve successful LV transduction in vivo ; even the maximal dose did not increase the risk of adverse events. Furthermore, the direct LV- ABCD1 injection displayed low immunogenicity. In addition, the effectiveness of intracerebral LV -ABCD1 injection has been preliminarily demonstrated while further investigation is needed. This study has been registered in the Chinese Clinical Trial Registry (https://www.chictr.org.cn/ , registration number: ChiCTR1900026649). [ABSTRACT FROM AUTHOR]

Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but their long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also up-regulated by lentiviral vectors exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 axis. [ABSTRACT FROM AUTHOR]

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field. [ABSTRACT FROM AUTHOR]

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. Synopsis: Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. Development of an in vitro phagocytosis assay, exploiting fluorescent virions and advanced imaging techniques, suitable to assess the susceptibility to phagocytosis of different LV versions in different experimental settings. Efficient tropism switch of LV from hepatocytes to liver endothelial cells in vitro and in vivo and shielding from phagocytosis. Reconstitution of FVIII activity in the normal range by a single intravenous administration of GP64-LV equipped with the endogenous endothelial-specific FVIII promoter. Proof-of-concept of stable long-term gene therapy for hemophilia A in both adult and newborn mice. Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. [ABSTRACT FROM AUTHOR]

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

Abstract 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.

Investigations to understand the function and control of the globin genes have led to some of the most exciting molecular discoveries and biomedical breakthroughs of the 20th and 21st centuries. Extensive characterization of the globin gene locus, accompanied by pioneering work on the utilization of viruses as human gene delivery tools in human hematopoietic stem and progenitor cells (HPSCs), has led to transformative and successful therapies via autologous hematopoietic stem-cell transplant with gene therapy (HSCT-GT). Due to the advanced understanding of the β-globin gene cluster, the first diseases considered for autologous HSCT-GT were two prevalent β-hemoglobinopathies: sickle cell disease and β-thalassemia, both affecting functional β-globin chains and leading to substantial morbidity. Both conditions are suitable for allogeneic HSCT; however, this therapy comes with serious risks and is most effective using an HLA-matched family donor (which is not available for most patients) to obtain optimal therapeutic and safe benefits. Transplants from unrelated or haplo-identical donors carry higher risks, although they are progressively improving. Conversely, HSCT-GT utilizes the patient's own HSPCs, broadening access to more patients. Several gene therapy clinical trials have been reported to have achieved significant disease improvement, and more are underway. Based on the safety and the therapeutic success of autologous HSCT-GT, the U.S. Food and Drug Administration (FDA) in 2022 approved an HSCT-GT for β-thalassemia (Zynteglo™). This review illuminates the β-globin gene research journey, adversities faced, and achievements reached; it highlights important molecular and genetic findings of the β-globin locus, describes the predominant globin vectors, and concludes by describing promising results from clinical trials for both sickle cell disease and β-thalassemia.

Polyglutamine disorders are a complex group of incurable neurodegenerative disorders caused by an abnormal expansion in the trinucleotide cytosine-adenine-guanine tract of the affected gene. To better understand these disorders, our dependence on animal models persists, primarily relying on transgenic models. In an effort to complement and deepen our knowledge, researchers have also developed animal models of polyglutamine disorders employing viral vectors. Viral vectors have been extensively used to deliver genes to the brain, not only for therapeutic purposes but also for the development of animal models, given their remarkable flexibility. In a time- and cost-effective manner, it is possible to use different transgenes, at varying doses, in diverse targeted tissues, at different ages, and in different species, to recreate polyglutamine pathology. This paper aims to showcase the utility of viral vectors in disease modelling, share essential considerations for developing animal models with viral vectors, and provide a comprehensive review of existing viral-based animal models for polyglutamine disorders. [ABSTRACT FROM AUTHOR]

Aim: The present study examined the protective potential of human adipose tissue-derived mesenchymal stem cells (hASCs) modified to overexpress alpha-1 antitrypsin (AAT), in a mouse model of the liver fibrosis. Background: For the treatment of end-stage liver diseases, cell therapy has emerged as a promising noninvasive alternative to liver transplantation. Mesenchymal stem cells (MSCs) are being evaluated due to their dual capabilities of promoting liver regeneration and modulating the pathogenic inflammation of the immune system. Methods: Liver fibrosis was induced in mice via the intraperitoneal injection of carbon tetrachloride (CCl4). MSCs were extracted from the human adipose tissue. After stemness confirmation, the cells were transduced with the lentiviruses containing the AAT gene, and then injected into the mice's tail vein. Fourteen days' post-transplantation, mice were sacrificed, and blood and tissue samples were collected for analysis. Important liver enzymes, including alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, and total bilirubin (TB), were measured. Histological studies were carried out using the hematoxylin and eosin (H&E), as well as Masson's trichrome (MT) staining. Results: Compared to hASCs, treatment with AAT-hASCs resulted in greater reductions in ALT, AST, ALP, and TB, as well as normalized albumin levels. AAT-hASCs promoted enhanced liver regeneration histologically, likely attributable to anti-inflammatory and anti-proteolytic properties of AAT. Conclusion: These findings indicate AAT-engineered hASCs as a promising cell-gene therapy candidate for further study in liver cirrhosis models. [ABSTRACT FROM AUTHOR]

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of major histocompatibility complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion, reduces handling, and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes.

This article discusses the development of a lentiviral vector-based gene therapy platform for the treatment of liver metastases (LMS). Liver metastases commonly arise from gastrointestinal tumors and are difficult to treat due to the immunosuppressive environment of the liver. The lentiviral vector delivers interferon-alpha (IFNα) to liver macrophages, which activates the immune system and reduces the growth of LMS. The study also found that combining gene-based IFNα delivery with monoclonal antibodies blocking CTLA-4 resulted in a strong therapeutic response. The use of lentiviral vectors in gene therapy has shown promise in various clinical applications and has the potential to be used in the treatment of LMS. [Extracted from the article]

Current specific treatments for mucopolysaccharidoses (MPSs) include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT). Both treatments are hampered by several limitations, including lack of efficacy on brain and skeletal manifestations, need for lifelong injections, and high costs. Therefore, more effective treatments are needed. Gene therapy in MPSs is aimed at obtaining high levels of the therapeutic enzyme in multiple tissues either by engrafted gene‐modified hematopoietic stem progenitor cells (ex vivo) or by direct infusion of a viral vector expressing the therapeutic gene (in vivo). This review focuses on the most recent clinical progress in gene therapies for MPSs. The various gene therapy approaches with their strengths and limitations are discussed. [ABSTRACT FROM AUTHOR]

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion ofmyeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin-cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bonemarrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients. [ABSTRACT FROM AUTHOR]

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo. [ABSTRACT FROM AUTHOR]

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber's amaurosis, epidermolysis bullosa, Duchenne's muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results. Gene therapy offers the possibility for overexpression of secretable enzymes that can be released and through uptake, allow cross-correction of affected cells. Here, we discuss some of the leukodystrophies that have demonstrated strong potential for gene therapy interventions, such as X-linked adrenoleukodystrophy (X-ALD), and metachromatic leukodystrophy (MLD), which have reached clinical application. We further discuss the advantages and disadvantages of ex vivo lentiviral hematopoietic stem cell gene therapy, an approach for targeting microglia-like cells or rendering cross-correction. In addition, we summarize ongoing developments in the field of in vivo administration of recombinant adeno-associated viral (rAAV) vectors, which can be used for direct targeting of affected cells, and other recently developed molecular technologies that may be applicable to treating leukodystrophies in the future. [ABSTRACT FROM AUTHOR]

Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL. [ABSTRACT FROM AUTHOR]

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in the aging population, with vascular endothelial growth factor (VEGF) playing a key role. Treatment with recombinant anti-VEGFs is the current standard of care; however, it is only effective for 1–2 months at a time and requires re-administration. Gene therapy could pave the way for stable, long-term expression of therapeutic anti-VEGF with a single dose, reducing the frequency of treatment and potentially improving clinical outcomes. As such, we have developed OXB-203, a lentiviral-based gene therapy encoding the anti-VEGF protein aflibercept. Aflibercept derived from OXB-203 exhibited comparable in vitro binding characteristics to VEGF as recombinant aflibercept. Furthermore, its biological potency was demonstrated by the equivalent inhibition of VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and tubule formation as recombinant aflibercept. In a rat choroidal neovascularization (CNV) model of nAMD, a single subretinal administration of OXB-203 reduced laser-induced CNV lesion areas analogous to an intravitreal bolus of recombinant aflibercept. Finally, in a head-to-head comparative study, aflibercept derived from OXB-203 was shown to be expressed at significantly higher levels in ocular tissues than from an AAV8-aflibercept vector following a single subretinal delivery to rats. These findings support the therapeutic potential of OXB-203 for the management of nAMD.

Lentiviral vectors (LVVs) play a critical role in gene delivery for ex vivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5 × 104 to 5 × 103 cells/cm2, delaying the transient transfection from 24 to 120 h post-seeding, reducing plasmid DNA to 167 ng/cm2, and adding 5 mM sodium butyrate 6 h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2 × 106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum both at small and large scale.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

Background and purpose: Chimeric antigen receptor T (CAR-T) cell technology has been widely used in the field of blood tumor treatment. Lentivirus transduction is a key link in the preparation of CAR-T cells, which is closely related to the quality of CAR-T cells. Therefore, the parameters involved in the lentivirus transduction process still need to be further optimized. This study aimed to investigate the effects of multiplicity of infection (MOI), incubation density, activation time before transduction and transduction system height on anti-CD30 CAR-T cells in lentiviral transduction T cells carrying CD30 antibody sequences, optimize the transduction conditions of CAR-T cells, and improve transduction efficiency and CAR-T cells function. Methods: Different MOI, incubation density, activation time before transduction and transduction system height were used to optimize the transduction of human peripheral blood-derived T cells, then proliferation capacity, transduction efficiency, cell viability and killing efficiency in vitro of anti-CD30 CAR-T cells were detected. Results: When the MOI was 1.00, 1.50 and 3.00, the transduction efficiency and effective cell number were significantly higher than those in the 0.00, 0.25 and 0.50 groups. When the incubation density was greater than 0.5×107 cells/mL, the incubation density had no significant effect on the transduction efficiency of T cells. The cell viability rate of the 72 h group was lower than 80%, which was lower compared with other groups significantly, the transduction efficiency of 24 and 48 h was significantly higher compared with the 0 h, 8 h and 16 h groups, and the expansion rate of CAR-T cells in the 48 h group was significantly higher compared with the 24 h group. The transduction efficiency and proliferation capacity of 0.16 mm in height of transduction system were significantly higher compared with the 0.53 mm group, however, had no significant effect on killing efficiency of CAR-T cells in vitro. Conclusion: Through the comprehensive evaluation of CAR-T cells function, the optimal transduction conditions for lentivirus include MOI of 1.00, incubation density of 1.0×107 cells/mL, activation before transduction for 48 h and transduction system height of 0.16 mm.

In cell and gene therapy, achieving the stable engraftment of an abundant and highly polyclonal population of gene-corrected cells is one of the key factors to ensure the successful and safe treatment of patients. Because integrative vectors have been associated with possible risks of insertional mutagenesis leading to clonal dominance, monitoring the relative abundance of individual vector insertion sites in patients’ blood cells has become an important safety assessment, particularly in hematopoietic stem cell-based therapies. Clinical studies often express clonal diversity using various metrics. One of the most commonly used is the Shannon index of entropy. However, this index aggregates two distinct aspects of diversity, the number of unique species and their relative abundance. This property hampers the comparison of samples with different richness. This prompted us to reanalyze published datasets and to model the properties of various indices as applied to the evaluation of clonal diversity in gene therapy. A normalized version of the Shannon index, such as Pielou’s index, or Simpson’s probability index is robust and useful to compare sample evenness between patients and trials. Clinically meaningful standard values for clonal diversity are herein proposed to facilitate the use of vector insertion site analyses in genomic medicine practice.

Sickle cell disease (SCD) is due to a mutation in the β-globin gene causing production of the toxic sickle hemoglobin (HbS; α2βS2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiRNA) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of the sickling phenotype, outperforming βAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

For gene therapies to become more accessible and affordable treatment options, process intensification is one possible strategy to increase the number of doses generated per batch of viral vector. Process intensification for lentiviral vector manufacturing can be achieved by enabling perfusion in the production bioreactor when applied in tandem with a stable producer cell line, allowing for significant expansion of cells and production of lentiviral vectors without the need for transfer plasmids. Tangential flow depth filtration was used to achieve an intensified lentiviral vector production by enabling perfusion to expand cell density and allow for continuous separation of lentiviral vectors from producer cells. Hollow-fiber depth filters made of polypropylene with 2- to 4-μm channels demonstrated high filter capacity, extended functional life, and efficient separation of lentiviral vectors from producer cells and debris when used for this intensified process. We anticipate that process intensification with tangential flow depth filtration at 200-L scale from a suspension culture can produce on the order of magnitude of 10,000 doses per batch of lentiviral vectors required for CAR T or TCR cell and gene therapy that would require approximately 2 × 109 transducing units per dose.

Mucopolysaccharidosis type II (MPSII) is a pediatric lysosomal storage disease caused by deficiencies in the IDS (iduronate-2-sulfatase) gene resulting in accumulation of glycosaminoglycans, multisystem disease, and profound neurodegeneration in severe forms. Although enzyme replacement therapy is available for somatic forms of disease, the inability of native IDS to pass the blood-brain barrier renders it ineffective for the brain. We previously demonstrated the short-term efficacy of a brain-targeted hematopoietic stem cell gene therapy approach to treat MPSII mice using lentiviral IDS fused to the blood-brain-barrier-crossing peptide ApoEII (IDS.ApoEII) in comparison with a lentivirus expressing native IDS and an unmanipulated bone marrow transplant. Here we evaluated the longevity of disease correction for 12–16 months following treatment. We observed sustained IDS enzyme activity in organs of long-term IDS.ApoEII-treated MPSII mice, similar to those analyzed 6 months post-treatment, with continued clearance of storage material in the brain and peripheral organs, maintained correction of astrogliosis, microgliosis, and correction of altered cytokines and chemokines. IDS.ApoEII also significantly reduced retinal atrophy, characteristic of MPSII. Overall, IDS.ApoEII resulted in systemic prevention of the MPSII phenotype, with no observed toxicity following treatment. This provides evidence of the sustained efficacy and safety of this treatment ahead of a recently opened clinical trial.

IntroductionRecombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation.MethodsIn this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter.Results and discussionStarting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Lysosomal Storage Disorders (LSDs) are a diverse group of inherited, monogenic diseases caused by functional defects in specific lysosomal proteins. The lysosome is a cellular organelle that plays a critical role in catabolism of waste products and recycling of macromolecules in the body. Disruption to the normal function of the lysosome can result in the toxic accumulation of storage products, often leading to irreparable cellular damage and organ dysfunction followed by premature death. The majority of LSDs have no curative treatment, with many clinical subtypes presenting in early infancy and childhood. Over two‐thirds of LSDs present with progressive neurodegeneration, often in combination with other debilitating peripheral symptoms. Consequently, there is a pressing unmet clinical need to develop new therapeutic interventions to treat these conditions. The blood–brain barrier is a crucial hurdle that needs to be overcome in order to effectively treat the central nervous system (CNS), adding considerable complexity to therapeutic design and delivery. Enzyme replacement therapy (ERT) treatments aimed at either direct injection into the brain, or using blood–brain barrier constructs are discussed, alongside more conventional substrate reduction and other drug‐related therapies. Other promising strategies developed in recent years, include gene therapy technologies specifically tailored for more effectively targeting treatment to the CNS. Here, we discuss the most recent advances in CNS‐targeted treatments for neurological LSDs with a particular emphasis on gene therapy‐based modalities, such as Adeno‐Associated Virus and haematopoietic stem cell gene therapy approaches that encouragingly, at the time of writing are being evaluated in LSD clinical trials in increasing numbers. If safety, efficacy and improved quality of life can be demonstrated, these therapies have the potential to be the new standard of care treatments for LSD patients. [ABSTRACT FROM AUTHOR]

Background and purpose: Chimeric antigen receptor T (CAR-T) cell technology has been widely used in the field of blood tumor treatment. Lentivirus transduction is a key link in the preparation of CAR-T cells, which is closely related to the quality of CAR-T cells. Therefore, the parameters involved in the lentivirus transduction process still need to be further optimized. This study aimed to investigate the effects of multiplicity of infection (MOI), incubation density, activation time before transduction and transduction system height on anti-CD30 CAR-T cells in lentiviral transduction T cells carrying CD30 antibody sequences, optimize the transduction conditions of CAR-T cells, and improve transduction efficiency and CAR-T cells function. Methods: Different MOI, incubation density, activation time before transduction and transduction system height were used to optimize the transduction of human peripheral blood-derived T cells, then proliferation capacity, transduction efficiency, cell viability and killing efficiency in vitro of anti-CD30 CAR-T cells were detected. Results: When the MOI was 1.00, 1.50 and 3.00, the transduction efficiency and effective cell number were significantly higher than those in the 0.00, 0.25 and 0.50  groups. When the incubation density was greater than 0.5×107 cells/mL, the incubation density had no significant effect on the transduction efficiency of T cells. The cell viability rate of the 72  h group was lower than 80%, which was lower compared with other groups significantly, the transduction efficiency of 24 and 48 h was significantly higher compared with the 0  h, 8  h and 16  h groups, and the expansion rate of CAR-T cells in the 48  h group was significantly higher compared with the 24 h group. The transduction efficiency and proliferation capacity of 0.16  mm in height of transduction system were significantly higher compared with the 0.53  mm group, however, had no significant effect on killing efficiency of CAR-T cells in vitro. Conclusion: Through the comprehensive evaluation of CAR-T cells function, the optimal transduction conditions for lentivirus include MOI of 1.00, incubation density of 1.0×107 cells/mL, activation before transduction for 48 h and transduction system height of 0.16 mm. [ABSTRACT FROM AUTHOR]

Gene-modification therapies are at the forefront of HIV-1 cure strategies. Chimeric antigen receptor (CAR)-T cells pose a potential approach to target infected cells during antiretroviral therapy or following analytical treatment interruption (ATI). However, there are technical challenges in the quantification of HIV-1-infected and CAR-T cells in the setting of lentiviral CAR gene delivery and also in the identification of cells expressing target antigens. First, there is a lack of validated techniques to identify and characterize cells expressing the hypervariable HIV gp120 in both ART-suppressed and viremic individuals. Second, close sequence homology between lentiviral-based CAR-T gene modification vectors and conserved regions of HIV-1 creates quantification challenges of HIV-1 and lentiviral vector levels. Consideration needs to be taken into standardizing HIV-1 DNA/RNA assays in the setting of CAR-T cell and other lentiviral vector-based therapies to avoid these confounding interactions. Lastly, with the introduction of HIV-1 resistance genes in CAR-T cells, there is a need for assays with single-cell resolution to determine the competence of the gene inserts to prevent CAR-T cells from becoming infected in vivo. As novel therapies continue to arise in the HIV-1 cure field, resolving these challenges in CAR-T-cell therapy will be crucial. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract Chimeric antigen receptor (CAR) T cell therapy, in which a patient’s own T lymphocytes are engineered to recognize and kill cancer cells, has achieved remarkable success in some hematological malignancies in preclinical and clinical trials, resulting in six FDA-approved CAR-T products currently available in the market. Once equipped with a CAR construct, T cells act as living drugs and recognize and eliminate the target tumor cells in an MHC-independent manner. In this review, we first described all structural modular of CAR in detail, focusing on more recent findings. We then pointed out behind-the-scene elements contributing to CAR expression and reviewed how CAR expression can be drastically affected by the elements embedded in the viral vector backbone.

A promising direction in the treatment of HIV infection is a gene therapy approach based on the insertion of antiviral genes aimed at inhibiting HIV replication into the genome of host cells. We obtained six constructs of lentiviral vectors with different arrangements of three antiviral genes: microRNAs against the CCR5 gene, the gene encoding the C-peptide, and the gene encoding the modified human TRIM5a protein. We found that despite containing the same genes, these vectors were produced at different titers and had different effects on cell viability, transduction efficiency, and expression stability. Comparative evaluation of the antiviral activity of three of the six developed vectors that showed stable expression was carried out using the continuous SupT1 lymphocytic cell line. All of the vectors protected cells from HIV infection: the viral load was several orders of magnitude lower than in control cells, and with one vector, complete cessation of virus growth in modified cells was achieved. [ABSTRACT FROM AUTHOR]

Gene therapy relies on the delivery of genetic material to the patient's cells in order to provide a therapeutic treatment. Two of the currently most used and efficient delivery systems are the lentiviral (LV) and adeno-associated virus (AAV) vectors. Gene therapy vectors must successfully attach, enter uncoated, and escape host restriction factors (RFs), before reaching the nucleus and effectively deliver the therapeutic genetic instructions to the cell. Some of these RFs are ubiquitously expressed in mammalian cells, while others are cell-specific, and others still are expressed only upon induction by danger signals as type I interferons. Cell restriction factors have evolved to protect the organism against infectious diseases and tissue damage. These restriction factors can be intrinsic, directly acting on the vector, or related with the innate immune response system, acting indirectly through the induction of interferons, but both are intertwined. The innate immunity is the first line of defense against pathogens and, as such cells derived from myeloid progenitors (but not only), are well equipped with RFs to detect pathogen-associated molecular patterns (PAMPs). In addition, some non-professional cells, such as epithelial cells, endothelial cells, and fibroblasts, play major roles in pathogen recognition. Unsurprisingly, foreign DNA and RNA molecules are among the most detected PAMPs. Here, we review and discuss identified RFs that block LV and AAV vector transduction, hindering their therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

Genetic deficiency of β-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.

Conjugating certain types of lentiviral vectors with targeting ligands can redirect the vectors to specifically transduce desired cell types. However, extensive genetic and/or biochemical manipulations are required for conjugation, which hinders applications for targeting lentiviral vectors for broader research fields. We developed envelope proteins fused with biotin-binding molecules to conjugate the pseudotyped vectors with biotinylated targeting molecules by simply mixing them. The envelope proteins fused with the monomeric, but not tetrameric, biotin-binding molecules can pseudotype lentiviral vectors and be conjugated with biotinylated targeting ligands. The conjugation is stable enough to redirect lentiviral transduction in the presence of serum, indicating their potential in in vivo . When a signaling molecule is conjugated with the vector, the conjugation facilitates transduction and signaling in a receptor-specific manner. This simple method of ligand conjugation and ease of obtaining various types of biotinylated ligands will make targeted lentiviral transduction easily applicable to broad fields of research.

Following the breakthrough of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in recent months and the incomplete efficiency of the currently available vaccines, development of more effective vaccines is desirable. Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models and are particularly suitable for mucosal vaccination, which is acknowledged as the most effective in reducing viral transmission. Here, we demonstrate that a single intranasal administration of a vaccinal lentiviral vector encoding a stabilized form of the original SARS-CoV-2 Spike glycoprotein induces full-lung protection of respiratory tracts and strongly reduces pulmonary inflammation in the susceptible Syrian golden hamster model against the prototype SARS-CoV-2. In addition, we show that a lentiviral vector encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P) prevents pathology and reduces infectious viral loads in lungs and nasal turbinates following inoculation with the SARS-CoV-2 Omicron variant. Importantly, an intranasal boost with LV::SBeta-2P improves cross-seroneutralization much better in LV::SBeta-2P-primed hamsters than in their counterparts primed with an LV-encoding Spike from the ancestral SARS-CoV-2. These results strongly suggest that an immune imprint with the original Spike sequence has a negative impact on cross-protection against new variants. Our results tackle the issue of vaccine effectiveness in people who have already been vaccinated and have vanished immunity and indicate the efficiency of LV-based intranasal vaccination, either as a single dose or as booster. [ABSTRACT FROM AUTHOR]

Platelets are anucleate blood cells that are shed from megakaryocytes (MKs) into the bloodstream to maintain hemostasis and promote wound healing after vascular injury. To carry out their functions, platelets become activated and release bioactive substances from their secretory granules. As alpha granules (αGs) in resting platelets store proteins and release them only after activation, the packaging of proteins into αGs is an attractive strategy to deliver therapeutic proteins. Here, we propose an adjustable model for targeting transgenic proteins to platelet αGs using third-generation self-inactivating lentiviral vectors. The vectors express from the murine platelet factor 4 promoter (mPf4P), restricting transgene expression to the MK lineage. For the delivery and retention of expressed proteins in αGs, proteins are fused to short peptide sorting signals derived from the human cytokine RANTES or from the transmembrane protein P-selectin. We demonstrate effective targeting of GFP to αGs of murine and human in vitro-differentiated MKs and murine platelets in vivo. Furthermore, interferon-α (IFNα), as a potentially therapeutic cytokine, was successfully delivered to and stored in murine platelets in vivo, was released after activation, and inhibited virus replication in vitro. Our vectors create possibilities for numerous applications in cell therapy utilizing platelets as carriers of therapeutic proteins.

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber’s amaurosis, epidermolysis bullosa, Duchenne’s muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results. Gene therapy offers the possibility for overexpression of secretable enzymes that can be released and through uptake, allow cross-correction of affected cells. Here, we discuss some of the leukodystrophies that have demonstrated strong potential for gene therapy interventions, such as X-linked adrenoleukodystrophy (X-ALD), and metachromatic leukodystrophy (MLD), which have reached clinical application. We further discuss the advantages and disadvantages of ex vivo lentiviral hematopoietic stem cell gene therapy, an approach for targeting microglia-like cells or rendering cross-correction. In addition, we summarize ongoing developments in the field of in vivo administration of recombinant adeno-associated viral (rAAV) vectors, which can be used for direct targeting of affected cells, and other recently developed molecular technologies that may be applicable to treating leukodystrophies in the future.

The application of retroviral vectors in the laboratory requires considerations that often go overlooked but are often easy to circumvent. Here, we discuss the relationship between the observed transduction efficiency of a cell population and per-cell viral insertions—and describe how differential cell-type susceptibilities can confound results. We consider the math underlying this problem and review an alternative approach to the commonly used “multiplicity of infection” (MOI) method of titering and using viral vectors in the biomedical research laboratory.

The most prevalent malignant bone tumor, osteosarcoma, affects the growth plates of long bones in adolescents and young adults. Standard chemotherapeutic methods showed poor response rates in patients with recurrent and metastatic phases. Therefore, it is critical to develop novel and efficient targeted therapies to address relapse cases. In this regard, RNA interference technologies are encouraging options in cancer treatment, in which small interfering RNAs regulate the gene expression following RNA interference pathways. The determination of target tissue is as important as the selection of tissue-specific promoters. Moreover, small interfering RNAs should be delivered effectively into the cytoplasm. Lentiviral vectors could encapsulate and deliver the desired gene into the cell and integrate it into the genome, providing long-term regulation of targeted genes. Silencing overexpressed genes promote the tumor cells to lose invasiveness, prevents their proliferation, and triggers their apoptosis. The uniqueness of cancer cells among patients requires novel therapeutic methods that treat patients based on their unique mutations. Several studies showed the effectiveness of different approaches such as microRNA, drug- or chemotherapy-related methods in treating the disease; however, identifying various targets was challenging to understanding disease progression. In this regard, the patient-specific abnormal gene might be targeted using genomics and molecular advancements such as RNA interference approaches. Here, we review potential therapeutic targets for the RNA interference approach, which is applicable as a therapeutic option for osteosarcoma patients, and we point out how the small interfering RNA method becomes a promising approach for the unmet challenge. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications. [ABSTRACT FROM AUTHOR]