Pseudotyped Lentiviral Vectors Research Articles

Selective, broad-spectrum antiviral photodynamic disinfection with dicationic imidazolyl chlorin photosensitizers

The COVID-19 pandemic exposes our vulnerability to viruses that acquire the ability to infect our cells. Classical disinfection methods are limited by toxicity. Existing medicines performed poorly against SARS-CoV-2 because of their specificity to targets in different organisms. We address the challenge of mitigating known and prospective viral infections with a new photosensitizer for antimicrobial photodynamic therapy (aPDT). Photodynamic inactivation is based on local oxidative stress, which is particularly damaging to enveloped viruses. We synthesized a cationic imidazolyl chlorin that reduced by > 99.999% of the percentage inhibition of amplification of SARS-CoV-2 collected from patients at 0.2 µM concentration and 4 J cm–2. Similar results were obtained in the prevention of infection of human ACE2-expressing HEK293T cells by a pseudotyped lentiviral vector exhibiting the S protein of SARS-CoV-2 at its surface. No toxicity to human epidermal keratinocytes (HaCaT) cells was found under similar conditions. aPDT with this chlorin offers fast and safe broad-spectrum photodisinfection and can be repeated with low risk of resistance.Graphical abstract

Establishment of High Titer Baboon Endogenous Retrovirus Envelope Pseudotyped Lentiviral Vector Production Protocol Focusing on Syncytium Formation and Exfoliation

Introduction Cytokine stimulation of hematopoietic stem cells (HSCs) for the expression of virus entry transporter is inevitable to obtain high transduction efficiency for VSVg envelope pseudotyped lentiviral vectors (VSVg-LVs). However, those stimulation induce apoptosis in HSCs derived from patients affected by DNA repair disorder, the establishment of novel transduction methods has been warranted. Sodium-dependent neutral amino acid transporter type-1 and type-2, which are the entry transporter for Baboon endogenous retrovirus (BaEV-Rless) envelope pseudotyped lentiviral vectors (BaEV-Rless-LVs), are highly expressed on the surface of HSCs in absence of cytokine stimulation. Therefore, BaEV-Rless-LVs could be an effective gene therapy tool to increase transduction in HSC with DNA repair-related mutations. However, production of high titer BaEV-Rless-LVs proved to be challenging compared to VSVg-LVs, as the titer of BaEV-Rless-LVs was one thousandth of VSVg-LVs. 293T cells transfected with helper plasmids, including BaEV-Rless plasmid, showed morphological changes such as syncytium formation and exfoliation, likely impairing cell viability and virus production. Therefore, for clinical applications, improved production of BaEV-Rless-LVs is mandatory. To this end, we modified the protocol to prevent the characteristic morphological changes, syncytium formation and exfoliation, which could be the causes of low virus titer. Methods In the attempt to increase viral production, we modified the VSVg-LVs "basic protocol" modified to produce BaEV-Rless-LVs: co-transfection of 0.44 µg of BaEV-Rless envelope plasmid, 1.464 µg of the self-inactivating HIV-1 derived vector encoding green fluorescent protein (GFP) under the control of spleen focus foamy virus promoter (SIN-HIV-SFFV-GFP), 0.336 μg of Rev regulatory plasmid and 0.74 μg of RRE plasmid to 293T cells per well of 6-well-plate, respectively. Virus titration was performed on 293T cells using serial dilution of the viral preparations and assessing 293T GFP+ cells using flow cytometry analysis. To investigate in detail the time-course of syncytium formation and exfoliation of transfected 293T cells, we used by time-lapse analysis to assess critical culture conditions and to guide us on how to further modify the protocol. Results Time-lapse analysis revealed that syncytium formation initiated at 8-hours post-transfection of the helper plasmids and terminated at 18-hours post-transfection. (Figure 1). Exfoliation started at 20-hours post-transfection, soon after completion of syncytium formation. As we hypothesized that the syncytium formation and exfoliation might be the cause of the low viral vector production, we modified three points of the basic protocol: the amount of BaEV-Rless plasmid, harvest timing of virus supernatant and use of poly-l-lysine (PLL) coated plate to avoid syncytium formation and exfoliation of 293T cells. First, to clarify whether the amount of BaEV-Rless plasmid was proportional to syncytium formation and virus titer, we changed the amounts of BaEV-Rless plasmid and compared titers. Interestingly, increasing 1.2-fold of the amount of plasmid raised the titer 3-fold despite syncytium formation. In contrast, even though smaller amounts of BaEV-Rless-plasmid did not induce syncytium formation and exfoliation, the titer of BaEV-Rless-LVs was reduced and/or undetectable. Next, we identified the optimal harvest time for viral titer. Harvest of viral supernatant at 48-hours post-transfection was associated with the highest titer despite completion of syncytium formation and exfoliation of 293T cells. Finally, we compared the titers with or without using PLL coated plate. Using PLL coated plate prevented exfoliation of transfected 293T cells and doubled the titer. Eventually, combination of using 1.2-folds of BaEV-Rless-plasmid, harvest of virus supernatant at 48-hours post-transfection and using PLL coated plate resulted in a 10-fold titer increase compared with the basic protocol (Figure 2). Conclusion Our novel high titer protocol for BaEV-Rless-LVs results in 10-fold titer increase compared to the basic protocol, and it could be utilized to develop clinical applications for DNA repair-related disorders. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Photodynamic disinfection of SARS-CoV-2 clinical samples using a methylene blue formulation.

The amplitude of the coronavirus disease 2019 (COVID-19) pandemic motivated global efforts to find therapeutics that avert severe forms of this illness. The urgency of the medical needs privileged repositioning of approved medicines. Methylene blue (MB) has been in clinical use for a century and proved especially useful as a photosensitizer for photodynamic disinfection (PDI). We describe the use of MB to photo-inactivate SARS-CoV-2 in samples collected from COVID-19 patients. One minute of treatment can reduce the percentage inhibition of amplification by 99.99% under conditions of low cytotoxicity. We employed a pseudotyped lentiviral vector (LVs) encoding the luciferase reporter gene and exhibiting the S protein of SARS-CoV-2 at its surface, to infect human ACE2-expressing HEK293T cells. Pre-treatment of LVs with MB-PDI prevented infection at low micromolar MB concentrations and 1 min of illumination. These results reveal the potential of MB-PDI to reduce viral loads in the nasal cavity and oropharynx in the early stages of COVID-19, which may be employed to curb the transmission and severity of the disease.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s43630-022-00202-6.

Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation.

ABSTRACTThe high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein’s function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions.IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.

Activating Natural Killer Cell Receptors, Selectins, and Inhibitory Siglecs Recognize Ebolavirus Glycoprotein

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus.

Towards Human Translation of Lentiviral Airway Gene Delivery for Cystic Fibrosis: A One-Month CFTR and Reporter Gene Study in Marmosets.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the luciferase (Luc), LacZ, and hCFTR transgenes was delivered into the trachea through a miniature bronchoscope. We examined whether a single-dose delivery of LV vector after LPC conditioning could increase levels of transgene expression in the trachea and lungs compared with control (phosphate-buffered saline [PBS]) conditioning. At 1 month, bioluminescence was detected in vivo in the trachea of three of the six animals within the PBS control group, compared with five of the six LPC-treated animals. When examined ex vivo there was weak evidence that LPC improves tracheal Luc expression levels. In the lungs, bioluminescence was detected in vivo in four of the six PBS-treated animals, compared with five of the six LPC-treated animals; however, bioluminescence was present in all lungs when imaged ex vivo. LacZ expression was predominantly observed in the alveolar regions of the lung. hCFTR was detected by qPCR in the lungs of five animals. Basal cells were successfully isolated and expanded from marmoset tracheas, but no LacZ-positive colonies were detected. There was no evidence of an inflammatory response toward the LV vector at 1 month postdelivery, with cytokines remaining at baseline levels. In conclusion, we found weak evidence that LPC conditioning improved gene transduction in the trachea, but not in the marmoset lungs. We also highlight some of the challenges associated with translational lung gene therapy studies in NHPs.

Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5′ end, to enable ribosomal entry from the 5′ 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.

Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector

BackgroundTo target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population. New MethodHere we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used. ResultsWe transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used. Comparison with existing methodThe optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements. ConclusionWe have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.

TEM-MM-101: A Phase I/IIa Dose Escalation Study Evaluating the Safety and Activity of Autologous CD34+ Enriched Hematopoietic Progenitor Cells Genetically Modified for Human Interferon-α2 in Multiple Myeloma Patients with Early Relapse after Intensive Front Line Therapy

BACKGROUND Multiple Myeloma (MM) is an incurable malignancy heralded by an immunosuppressive tumor microenvironment (TME). Therapeutic efforts to reprogram the TME and release innate and adaptive immunity may synergize with current anti-MM drugs, deepen responses and ultimately prolong long-term survival. We have developed an ex vivo gene therapy approach that locally delivers interferon-alpha (IFNa) into the TME through the Tie2+ myeloid progeny of transplanted gene-modified hematopoietic stem and progenitor cells (HSPCs). Preclinical studies in the immunocompetent Vk*Myc murine model as well as in the hematochimeric xenotransplantation setting demonstrate that IFNa gene therapy has anti-MM effect and may subvert the immunosuppressive MM TME, acting both on myeloma cells as well as on the immune infiltrate. Additional toxicology data, demonstrates that a mixture of genetically modified and unmodified CD34+ cells can be transplanted safely. We have therefore progressed into the clinic at Ospedale San Raffaele in Milan, Italy, with our gene therapy approach (Temferon). METHODS The TEM-MM-101 study recruits patients with MM in early relapse (within 18 months) after intensive front line therapy defined as triplet induction (at least 3 cycles including a proteasome inhibitor and/or immunomodulatory drug [IMID]) followed by single or double autologous stem cell transplantation, or, triplet or quadruplet induction including at least a proteasome inhibitor and an IMID, followed by consolidation/maintenance treatment. Three cohorts of 3 patients will be included in the study. Patients will be dosed sequentially in each cohort with data review by an independent data monitoring committee before dose escalation to the next cohort. Key eligibility criteria include achieving at least a VGPR after second line salvage therapy, age 18-70 years, ECOG <2 and Karnofsky >70%, adequate cardiac, renal, hepatic and pulmonary function, and willingness to use appropriate contraception if relevant. Important exclusion criteria include the presence of active autoimmune disease or ineligibility for autologous bone marrow transplant. After confirmation of eligibility, patients undergo autologous peripheral blood HSPC mobilization with lenograstim and plerixafor, before undergoing apheresis. After purification, CD34+ cells undergo transduction with a third-generation, vesicular stomatitis virus-G pseudo-typed lentiviral vector driving myeloid-specific IFNa2 expression. Patients will be offered maintenance treatment from successful HSPC collection until Temferon release. A reduced intensity conditioning regimen is administered at Day -2 followed by ASCT and administration of Temferon at Day 0. Following hematological recovery, patients will be discharged from the Transplant Unit with regular outpatient follow up. After Day +100 from Temferon infusion, patients will resume anti-myeloma consolidation/maintenance treatment according to best clinical practice, unless there is Temferon engraftment and absence of disease on Day +100 evaluations, documented by stringent complete response (sCR) according to the IMWG response criteria, imaging (PET, MRI) negativity, and bone marrow minimal residual disease < 10-4 by multi-parameter flow cytometry, to be confirmed by next generation sequencing. If these response criteria are met on Day +100, patients may stay off maintenance treatment, as long as MRD≤10-5 documented on bone marrow aspirates performed every 3 months, starting from the Day +180 visit. The primary endpoints for this study are: Engraftment of Temferon between Day +30 and Day +100The proportion of patients achieving hematologic recovery by Day +30 from ASCTSafety of Temferon (short-term tolerability of Temferon; stable blood counts and absence of cytopenias > grade 2, unless related to myeloma progression or concomitant medications; absence of systemic IFNa toxicity; absence of Replication Competent Lentivirus; absence of hematologic malignancy that is distinct from progression of the primary neoplasm) The study is actively recruiting patients in Italy. In parallel, a study using Temferon in patients with glioblastoma multiforme with a similar study design and approach (TEM-GBM-001) is also in progress with the first cohort fully recruited. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Mazzoleni:Genenta Science: Employment, Equity Ownership. Russo:Genenta Science: Consultancy, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial..

Establishment of an Alphavirus-Specific Neutralization Assay to Distinguish Infections with Different Members of the Semliki Forest complex.

Background: Alphaviruses are transmitted by arthropod vectors and can be found worldwide. Alphaviruses of the Semliki Forest complex such as chikungunya virus (CHIKV), Mayaro virus (MAYV) or Ross River virus (RRV) cause acute febrile illness and long-lasting arthralgia in humans, which cannot be clinically discriminated from a dengue virus or Zika virus infection. Alphaviruses utilize a diverse array of mosquito vectors for transmission and spread. For instance, adaptation of CHIKV to transmission by Aedes albopictus has increased its spread and resulted in large outbreaks in the Indian Ocean islands. For many alphaviruses commercial diagnostic tests are not available or show cross-reactivity among alphaviruses. Climate change and globalization will increase the spread of alphaviruses and monitoring of infections is necessary and requires virus-specific methods. Method: We established an alphavirus neutralization assay in a 384-well format by using pseudotyped lentiviral vectors. Results: MAYV-specific reactivity could be discriminated from CHIKV reactivity. Human plasma from blood donors infected with RRV could be clearly identified and did not cross-react with other alphaviruses. Conclusion: This safe and easy to use multiplex assay allows the discrimination of alphavirus-specific reactivity within a single assay and has potential for epidemiological surveillance. It might also be useful for the development of a pan-alphavirus vaccine.

Astrocyte-specific DJ-1 overexpression protects against rotenone-induced neurotoxicity in a rat model of Parkinson's disease

DJ-1 is a redox-sensitive protein with several putative functions important in mitochondrial physiology, protein transcription, proteasome regulation, and chaperone activity. High levels of DJ-1 immunoreactivity are reported in astrocytes surrounding pathology associated with idiopathic Parkinson's disease, possibly reflecting the glial response to oxidative damage. Previous studies showed that astrocytic over-expression of DJ-1 in vitro prevented oxidative stress and mitochondrial dysfunction in primary neurons. Based on these observations, we developed a pseudotyped lentiviral gene transfer vector with specific tropism for CNS astrocytes in vivo to overexpress human DJ-1 protein in astroglial cells. Following vector delivery to the substantia nigra and striatum of adult Lewis rats, the DJ-1 transgene was expressed robustly and specifically within astrocytes. There was no observable transgene expression in neurons or other glial cell types. Three weeks after vector infusion, animals were exposed to rotenone to induce Parkinson's disease-like pathology, including loss of dopaminergic neurons, accumulation of endogenous α-synuclein, and neuroinflammation. Animals over-expressing hDJ-1 in astrocytes were protected from rotenone-induced neurodegeneration, and displayed a marked reduction in neuronal oxidative stress and microglial activation. In addition, α-synuclein accumulation and phosphorylation were decreased within substantia nigra dopaminergic neurons in DJ-1–transduced animals, and expression of LAMP-2A, a marker of chaperone mediated autophagy, was increased. Together, these data indicate that astrocyte-specific overexpression of hDJ-1 protects neighboring neurons against multiple pathologic features of Parkinson's disease and provides the first direct evidence in vivo of a cell non-autonomous neuroprotective function of astroglial DJ-1.

Role of Basal Cells in Producing Persistent Lentivirus-Mediated Airway Gene Expression.

Cystic fibrosis (CF) lung disease is an ideal candidate for a genetic therapy. It has been shown previously that preconditioning with lysophosphatidylcholine (LPC) prior to lentiviral (LV) vector delivery results in long-term in vivo gene expression in the airway epithelium of CF mice. It was hypothesized that this outcome is largely due to transduction of airway basal cells that in turn pass the transgene onto their progeny. The aim of these studies was to confirm if the in vivo delivery of a human immunodeficiency virus type 1 (HIV-1) vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped LV vector following LPC airway conditioning results in transduction of mouse airway basal cells in situ and if the transgene is passed onto their progeny. Additionally, the study sought to determine the efficiency of in vitro transduction of human airway basal cells. First, normal mouse nasal airways were pretreated with LPC prior to delivery of a HIV-1 VSV-G pseudotyped LV vector carrying a LacZ marker gene (LV-LacZ). An epithelial ablation model utilizing polidocanol was then used to demonstrate that clonal outgrowth of linear and spotted clusters of transgene expressing ciliated, basal, and goblet cells occurs following transduction of basal cells. Second, human basal cells were cultured from primary bronchial epithelial cells, with identity confirmed by keratin 5 staining. High levels of transgene expression were found following LV-LacZ transduction. This study demonstrates the ability of the vector delivery protocol to transduce mouse airway basal cells, the LV vector to transduce human basal cells, and the likely role of these cells in maintaining long-term gene expression. These findings support and further develop the potential of LV gene transfer for persistent correction of CF airway disease.

Gene therapy for Cystic Fibrosis: Improved delivery techniques and conditioning with lysophosphatidylcholine enhance lentiviral gene transfer in mouse lung airways

ABSTRACT Purpose/Aim: Cystic fibrosis (CF) is the most common, fatal recessive genetic disease among the Caucasian population. Gene therapy has the potential to treat CF long term, however physiological barriers can prevent VSV-G pseudotyped lentiviral (LV) vectors from efficiently accessing the relevant receptors on the basolateral membrane of airway epithelial cells. The aims of this experiment were to use our new dose delivery techniques to determine whether conditioning the mouse lung conducting airways with lysophosphatidylcholine (LPC) improves the level of airway gene expression. Materials and Methods: Anaesthetised normal C57Bl/6 mice were intubated with an endotracheal cannula to non-invasively facilitate airway access. The airways were conditioned with 0.1% LPC, 0.3% LPC, or PBS (control) instilled via the ET tube. One hour later a VSV-G pseudotyped LV vector carrying the LacZ transgene was delivered. LacZ expression was measured by X-gal staining of the excised lungs 3 months after gene delivery. Results: Endotracheal intubation enabled precise dose delivery to the trachea and conducting airways. The cartilaginous airways of the groups conditioned with 0.1% and 0.3% LPC contained significantly larger numbers of LacZ positive cells compared to the PBS control group. In the LPC conditioned groups the majority of cell transduction was in ciliated epithelial cells. Conclusion: LPC conditioning prior to LV vector delivery, substantially enhanced the level of conducting airway gene expression after a single gene vector delivery. These results extend the previously established effectiveness of this protocol for producing gene expression in the nasal airways to the lung airways, the primary site of deleterious pathophysiology in CF individuals.

The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain

Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.

Protection Against H7 Subtype Influenza Virus Infection in Mice by Passive Transfer of Neutralizing Monoclonal Antibody.

H7 subtype influenza viruses pose serious threats to both the poultry industry and public health. Recent human infections of avian H7N9 influenza viruses with substantial morbidity and mortality have raised concerns about this virus becoming a potential pandemic pathogen. Neutralizing antibodies have been proven to be highly effective in blocking influenza virus infections. In this study, in order to develop an antibody-based immunoprophylaxis against H7 subtype influenza virus, we first generated a neutralizing monoclonal antibody (MAb) by using a pseudotyped lentiviral vector carrying the hemagglutinin protein of H7 subtype influenza virus. In vitro studies demonstrated that this neutralizing MAb completely inhibited the infection of an H7 subtype influenza virus to cells. The protective efficacy of this MAb was then further tested in a mouse model. It was shown that passive immunization of this MAb protected mice from local virus challenge. Results of the current study lay a foundation for the development of neutralizing MAb-mediated prophylactic strategies to combat human H7 influenza virus infections.

Nipah virus fusion protein: Importance of the cytoplasmic tail for endosomal trafficking and bioactivity

Nipah virus (NiV) is a highly pathogenic paramyxovirus which encodes two surface glycoproteins: the receptor-binding protein G and the fusion protein F. As for all paramyxoviruses, proteolytic activation of the NiV-F protein is an indispensable prerequisite for viral infectivity. Interestingly, proteolytic activation of NiV-F differs principally from other paramyxoviruses with respect to protease usage (cathepsins instead of trypsin- or furin-like proteases), and the subcellular localization where cleavage takes place (endosomes instead of Golgi or plasma membrane). To allow efficient F protein activation needed for productive virus replication and cell-to-cell fusion, the NiV-F cytoplasmic tail contains a classical tyrosine-based endocytosis signal (Y525RSL) that we have shown earlier to be needed for F uptake and proteolytic activation. In this report, we furthermore revealed that an intact endocytosis signal alone is not sufficient for full bioactivity. The very C-terminus of the cytoplasmic tail is needed in addition. Deletions of more than four residues did not affect F uptake or endosomal cleavage but downregulated the surface expression, likely by delaying the intracellular trafficking through endosomal-recycling compartments. Given that the NiV-F cytoplasmic tail is needed for timely and correct intracellular trafficking, endosomal cleavage and fusion activity, the influence of tail truncations on NiV-mediated cell-to-cell fusion and on pseudotyping lentiviral vectors is discussed.

189. Airway Pre-Treatment Enhances Mouse Lung Lentiviral Reporter Gene Expression

Cystic fibrosis (CF) is the most common, fatal genetic disease among the Caucasian population. VSV-G pseudotyped lentiviral (LV) gene vectors have the potential to treat CF long-term, however physiological barriers prevent efficient vector access to ciliated and basal (stem) cells residing on the basement membrane. We have previously demonstrated the effectiveness of our lysophosphatidylcholine (LPC) pre-treatment and LV vector dosing technique for producing ciliated and basal cell transduction in mouse nasal airways.

534. Development of Innovative Scalable Protocols for the Purification of Lentiviral Vectors Pseudotyped With GaLV-TR or Mutated Measles Virus Glycoproteins

Lentiviral (LV) vectors are powerful tools for gene transfer and genome editing in dividing and non-dividing cells but their clinical application requires complex and scalable processes of production which are not yet developed for all possible LV pseudotypes used in research. The ability of pseudotyping LV by different glycoproteins (gp) allows targeting of different tissues and types of cells. Modified Gibbon Ape Leukaemia Virus (GaLV-TR) gp pseudotyped LV are interesting to transduce specifically hematopoietic stem cells (HSC). Mutated measles virus gp engineered with single chain Fv can be redirected to specific cell surface receptors such as MHC Class II antigens (MV-CMH-II) enabling the transduction of dendritic cells. However, reliable and efficient purification schemes for these pseudotypes have not yet been developed. Here we report the development of a new upstream process (USP) protocol and for the first time novel downstream process (DSP) protocols allowing high yield production of stable and infectious LV-GaLV-TR and LV-MV-CMH-II particles. We identified critical conditions in chromatographic and tangential flow filtration (TFF) steps for preserving the infectivity/functionality of lentiviral vectors during purification. This was done by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and secondly by defining the optimal order of these steps. A three-step process was optimized for LV-GALV-TR purification consisting of one TFF, and two chromatographic steps (ion-exchange and size exclusion chromatography, permitting recoveries of >50% (ip – infectious particles). With this process, purified GaLV pseudotyped LV vectors enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin peptide, whereas in the same conditions non-purified vector transduced only 9% of the cells. A comparable, but simplified purification protocol was developed for LV-MV-CMH-II vector in a two step protocol (TFF, size exclusion chromatography) leading to yields of 60% (ip). Our protocols will allow for the first time the purification of various LV pseudotypes that are biologically-active, stable and with sufficient recovery in the perspective of preclinical studies and clinical applications.

Role of Phosphatidylserine Receptors in Enveloped Virus Infection

We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.