Lentiviral Vector Particles Research Articles

Exosomes with Engineered Brain Derived Neurotrophic Factor on Their Surfaces Can Proliferate Menstrual Blood Derived Mesenchymal Stem Cells: Targeted Delivery for a Protein Drug.

Despite the efficacy of brain derived neurotrophic factor (BDNF) in neuro-regenerative medicine, it can't pass the blood-brain barrier. Recently, exosomes have been harnessed for targeted delivery of therapeutics into brain. Given these facts, an engineered exosome capable of BDNF expression on the surface would be an amenable tool for drug delivery. The BDNF gene was cloned into a plex-lamp lentiviral vector and virus particles were packaged using the Torano method. HEK293T cells were transduced by the purified viruses to produce and purify recombinant exosomes displaying the fusion protein on their surfaces. Western blot, Zeta sizer, TEM, and ELISA methods were used for exosome characterization. The effect of engineered exosomes on menstrual blood-derived mesenchymal stem cells (Mens-MSCs) proliferation was evaluated by cell counting assay, MTT assay, and qPCR on the bcl2 and nestin genes. Approximately, 1.8 × 108 TdU/ml of the viral particles was purified from the transfected cells and transduced into the HEK293T. Western blot and ELISA methods confirmed the surface display of the LAMP-BDNF fusion. TEM graphs and Zeta sizer results confirmed the morphology and the size of purified exosomes. Treatment of Mens-MSCs with the targeted exosomes augmented the expression level of bcl2 and nestin genes, increased the cell proliferation, and elevated the cell number. Chimeric BDNF on the exosome surface could retain its biological activity and elevate the expression of bcl2 and nestin genes. Moreover, these exosomes are capable of elevating the Mens-MSCs proliferation.

Auto-transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy.

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7-6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

Digital Twin for Continuous Production of Virus-like Particles toward Autonomous Operation.

Lentiviral vector and virus-like particle (VLP) manufacturing have been published in fed-batch upstream and batch downstream modes before. Batch downstream and continuous upstream in perfusion mode were reported as well. This study exemplifies development and validation steps for a digital twin combining a physical-chemical-based mechanistic model for all unit operations with a process analytical technology strategy in order to show the efforts and benefits of autonomous operation approaches for manufacturing scale. As the general models are available from various other biologic manufacturing studies, the main step is model calibration for the human embryo kidney cell-based VLPs with experimental quantitative validation within the Quality-by-Design (QbD) approach, including risk assessment to define design and control space. For continuous operation in perfusion mode, the main challenge is the efficient separation of large particle manifolds for VLPs and cells, including cell debris, which is of similar size. Here, innovative tangential flow filtration operations are needed to avoid fast blocking with low mechanical stress pumps. A twofold increase of productivity was achieved using simulation case studies. This increase is similar to improvements previously described for other entities like plasmid DNAs, monoclonal antibodies (mAbs), and single-chain fragments of variability (scFv) fragments. The advantages of applying a digital twin for an advanced process control strategy have proven additional productivity gains of 20% at 99.9% reliability.

Abstract 48: T cell-specific in vivo transduction with preclinical candidate KLN-1010 generates BCMA directed CAR T cells with potent anti-multiple myeloma activity

Abstract Introduction: Multiple Myeloma (MM) is the second most diagnosed hematologic malignancy and remains an incurable disease. KLN-1010 is a novel treatment for MM currently in preclinical development that uses our iGPSTM platform, an envelope-modified lentiviral vector particle engineered for specific and efficient in vivo gene delivery, to transduce T cells. T cells modified in vivo by KLN-1010 express a fully human anti-BCMA CAR following a single IV injection without the need for additional treatments or conditioning chemotherapy. Results: Human T cells exposed to KLN-1010 in vitro resulted in transduction and expansion of anti-BCMA CAR T cells in a concentration dependent manner. The resulting CAR T cells were highly avid and specific to BCMA-expressing cell lines. KLN-1010 treatment of preclinical mouse models resulted in T cell-specific CAR expression with no evidence of off-target modification including in the lung and liver, except phagocytes. A varied CAR T cell lineage composition was observed that included effector and memory CD4 and CD8 T cells. A single dose of KLN-1010 caused potent anti-tumor efficacy and complete tumor regression in a stringent model that requires high dose levels of ex vivo-cultured CAR T cells similar to those commercially available. When the anti-BCMA CAR molecules used in KLN-1010 are compared in preclinical models to other clinically relevant CARs, we observed superior tumor control by the KLN-1010 anti-BCMA CAR molecules. Clinical application of iGPS technology was modeled in non-human primates (NHP) using an NHP T cell-targeting particle that expresses an anti-CD20 CAR. CAR T cell activity, assessed by B cell depletion, and tolerability were evaluated without prior lymphodepleting chemotherapy across a 10x dose range. Rapid clearance of the iGPS particles from the blood was observed at all dose levels. Even at a low dose of 1.3e8 IU/kg, potent and durable CAR T cell activity was observed as evidenced by complete B cell depletion lasting over two months. Multiple CAR T cell expansions and contractions were observed during this period. The treatment at all dose levels was highly tolerable with no observed toxicities, cytokine release syndrome, or neurotoxicity. Conclusions: These data demonstrate that KLN-1010 is safe and efficacious in preclinical models and may offer greater accessibility than other therapeutic modalities to address an unmet medical need in MM. Without requirements of ex vivo manufacturing, lymphodepletion and inpatient treatment that restrict patient access to therapy, the iGPS technology is well poised to provide significant clinical benefit for a multitude of indications as an off-the-shelf, single dose treatment. Citation Format: Emily T. Beura, Howard J. Latimer, Denise Wong, Jenifer Obrigewitch, Alyson J. Warr, Shirley Dong, Matty Ariel, Josue Figueroa, Joseph Figueroa, Connor S. Dobson, Sanath K. Janaka, Alexander J. Najibi, Maggie Lau, Shannon Contrastano, Kevin Friedman. T cell-specific in vivo transduction with preclinical candidate KLN-1010 generates BCMA directed CAR T cells with potent anti-multiple myeloma activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 48.

In Vivo Generation of Functional CD19 CAR-T Cells Using a Serum-Stable CD3-Targeted Lentiviral Vector

The high cost and complexity of CAR-T therapy has spurred efforts to generate CAR-T cells directly within patients via systemic infusion of serum-stable lentiviral vector particles that target T cells in situ for CAR transgene delivery. To ensure effective in vivo delivery, these vectors need to show resistance to neutralization by human serum, demonstrate transduction specificity for T cells, and support subsequent expansion of CAR-T cells. Lentiviral vectors (LV) pseudotyped with VSV G-glycoprotein (G-WT) transduce target cells via the LDL receptor (LDLR), which is ubiquitously expressed at variable levels and is upregulated on activated T cells. G-WT LVs are widely used to generate therapeutic CAR-T cells ex vivo, following purification and ex vivo activation of the T cells. To generate LVs capable of targeting circulating T cells in vivo, we utilized rational engineering of the VSV-G glycoprotein to design a G-CD3. This modified G protein avoids LDLR recognition, incorporates a CD3-specific single-chain antibody for targeted recognition of T cells, and minimizes LV particle inactivation upon exposure to human serum. G-CD3 LVs were shown to target and transduce resting T cells in peripheral blood mononuclear cell (PBMC) cultures with very high efficiency. The G-CD3 LVs powerfully activated T cells, evident from their increased CD25 expression, and the infection was not hindered in the presence of fresh, complement-active human serum (25%). Additionally, G-CD3 LVs efficiently transduced T cells in fresh human whole blood without apparent off-target transduction of non-T cell lineages. In vivo evaluation of G-CD3 LV efficiency was performed in humanized NSG mice by intravenous (IV) or intraperitoneal (IP) delivery of G-CD3 LVs encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3 LV led to in vivo CAR T cell generation with peak levels of 17% of total circulating human CD45+ cells on day 21 post vector administration. The in vivo generated CD19-CAR-T cells were fully functional as evidenced by the disappearance of endogenous human CD19+ B cells from peripheral blood. Tissue analysis of spleen, bone marrow, and liver at the time of sacrifice (day 39 post LV injection), showed anti-CD19 CAR-T in all three tissues accompanied by an absence or dramatic reduction in tissue-resident human CD19+ B cells. In contrast, CAR-T cells were not detected at any time point in humanized NSG mice injected with G-WT LV, regardless of whether the virus was administered IV or IP. In summary, these data highlight the potential applicability of fully retargeted, serum stable LVs for in vivo gene delivery or genome editing, offering promising prospects for future human gene therapy applications.

Emetine in Combination with Chloroquine Induces Oncolytic Potential of HIV-1-Based Lentiviral Particles.

Chloroquine and Emetine are drugs used to treat human parasitic infections. In addition, it has been shown that these drugs have an antiviral effect. Both drugs were also found to cause a suppressive effect on the growth of cancer cells of different origins. Here, using the replication-deficient HIV-1-based lentiviral vector particles, we evaluated the ability of the combination of these drugs to reduce viral transduction efficiency. We showed that these drugs act synergistically to decrease cancer cell growth when added in combination with medium containing lentiviral particles. We found that the combination of these drugs with lentiviral particles decreases the viability of treated cells. Taken together, we state the oncolytic potential of the medium containing HIV-1-based particles provoked by the combination of Chloroquine and Emetine.

Steric exclusion chromatography of lentiviral vectors using hydrophilic cellulose membranes

Enveloped viral vectors like lentiviral vectors pose purification challenges due to their low stability. A gentle purification method is considered one of the major bottlenecks for lentiviral vector bioprocessing. To overcome these challenges, a promising method is steric exclusion chromatography which has been used to purify a variety of target molecules. In this study, we successfully identified optimal process parameters for steric exclusion chromatography to purify lentiviral vectors. Lentiviral vector particle recoveries and infectious recoveries of 86% and 88%, respectively, were achieved. The process parameters optimal for steric exclusion chromatography were determined as follows: polyethylene glycol with a molecular weight of 4000 Da, a polyethylene glycol concentration of 12.5%, and a flow rate of 7 mL⋅min−1 using 5 layers of stabilized cellulose membranes as a stationary phase. High protein and dsDNA removal of approximately 80% were obtained. The remaining polyethylene glycol concentration in the eluate was determined. We defined the maximum loading capacity as 7.5 × 1012 lentiviral particles for the lab device used and provide deeper insights into loading strategies. Furthermore, we determined critical process parameters like pressure. We demonstrated in our experiments that steric exclusion chromatography is a gentle purification method with high potential for fragile enveloped viral vectors as it yields high recoveries while efficiently removing impurities.

Clinical grade lentiviral vector purification and quality control requirements.

Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand for clinical-grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain a sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the lentiviral vector particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of lentiviral vector manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of lentiviral vectors in scalable processes.

Liver-Directed Gene Therapy for Hemophilia B with Immune Stealth Lentiviral Vectors

Gene therapy is rapidly gaining renewed interest and is emerging as a realistic treatment option for several genetic and acquired diseases. Underlying this success is the development of improved gene transfer vectors. Among them, lentiviral vectors (LV) are emerging as versatile vehicles of relatively large capacity for stable transgene integration in the genome of target cells. Over the past years, we developed LV that achieve stable transgene expression in the liver and establish correction of hemophilia, in mouse and dog models of the disease, upon systemic administration. These LV are designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We previously evaluated portal vein administration of LV expressing canine factor IX (FIX) in 3 adult hemophilia B dogs and reported stable multiyear reconstitution of FIX activity up to 1 % of normal. We have more recently treated 4 dogs as pups (2-4 months of age) by peripheral vein administration of escalating doses of LV (5 to 10e9 T.U./kg) expressing a hyper-functional canine FIX and achieved reconstitution of FIX activity up to 32% of normal at the highest dose tested. We then modified the vector envelope by changing the protein composition of the producer cell plasma membrane through genome editing to reduce immunogenicity of LV particles. First, we performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all class-I major histocompatibility complexes (MHC-I) to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. These MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with autologous monocytes exposed to LV, from several healthy donors, suggesting that conventional MHC-bearing LV may trigger allogeneic immune responses. Secondly, we generated LV with increasedlevels of CD47, a phagocytosis inhibitor, on the vector surface (CD47high LV), which show substantially decreased uptake by human macrophages in vitro . In order to evaluate the role of CD47 in LV biodistribution upon in vivo administration we took advantage of the non-obese diabetic (NOD) mouse model whose SIRPα (the CD47 receptor) is known to have high affinity for the human CD47. In this setting, CD47 proved to be a key player in reducing phagocytosis of LV, decreasing the inflammatory cytokine response following their administration, and increasing hepatocyte transduction and FIX output. We have now administered MHC-free or MHC-free/CD47high LV to 6 non-human primates (NHP, 3 for each LV version). We chose Macaca nemestrina as recipient, because of the lower restriction to HIV infection than other NHP species. Administration of 7.5e9 T.U./kg LV via peripheral vein was well tolerated, without significant elevation of serum aminotransferases or body temperature and only caused a transient self-limiting leukopenia. Remarkably, human-specific FIX activity reached up to 300% of normal and was nearly 3-fold higher in the CD47high-LV treated animals, showing a much more favorable LV dose-response than observed in mice and dogs. Upon necropsy, we measured vector copies in liver, spleen and major organs of treated animals and found between 0.5 and 1.5 LV copies in the liver accounting for 80-90% of all the retrieved LV copies, showing selective targeting and efficient gene transfer to the liver by LV in NHP. Overall, our studies support the efficacy and safety of these immune-stealth LV in NHP and position them to address some of the outstanding challenges in liver-directed gene therapy for hemophilia and conceivably other diseases. DisclosuresLiu:Bioverativ: Employment, Other: Shareholder. Peters:Bioverativ: Employment, Other: Shareholder.

The Engineered MARCH8-Resistant Vesicular Stomatitis Virus Glycoprotein Enhances Lentiviral Vector Transduction.

Lentiviral vectors are one of the most commonly used viral delivery systems for gene therapy. Vesicular stomatitis virus-G envelope glycoprotein (VSV G)-pseudotyped lentiviral vectors have been widely used in clinical studies for treatment of virus infections and genetic deficient diseases. However, the efficiency of lentiviral vector transduction has been long recognized as a limiting factor in clinical gene therapy application, especially in transducing hematopoietic stem cells. MARCH8 (membrane-associated RING-CH 8), an E3 ubiquitin ligase, has been reported to target and downregulate VSV G. Results in this study show that MARCH8 induces ubiquitination and lysosome degradation of VSV G, and knockout of MARCH8 in virus-producing cells increases lentiviral vector transduction by elevating the level of VSV G protein. We then engineered VSV G mutant that has the lysine residues in the cytoplasmic domain substituted for arginine, and showed that this G mutant resists degradation by MARCH8, and allows the enhancement of transduction efficiency of lentiviral vector particles than the parental VSV G protein. This engineered VSV G mutant thus further advances the lentiviral vector system as a powerful tool in gene therapy.

Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

The stability of envelope-pseudotyped lentiviral vectors

Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the ‘Reduction Ratio.’ This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector’s envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2–110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.

Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography

Lentiviral vectors (LVs) are a popular gene delivery tool in cell and gene therapy and they are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptor (CAR) in CAR-T cell therapies. Extensive process and product characterization are required in manufacturing virus-based gene vectors to better control batch-to-batch variability. However, it has been an ongoing challenge to make quantitative assessments of LV product because current analytical tools often are low throughput and lack robustness and standardization is still required. This paper presents a high-throughput and robust physico-chemical characterization method that directly assesses total LV particles. With simple sample preparation and fast elution time (6.24 min) of the LV peak in 440 mM NaCl (in 20 mM Tris-HCl [pH 7.5]), this ion exchange high-performance liquid chromatography (IEX-HPLC) method is ideal for routine in-process monitoring to facilitate the development of scalable and robust LV manufacturing processes. Furthermore, this HPLC method is suitable for the analysis of all in-process samples, from crude samples such as LV supernatants to final purified products. The linearity range of the standard curve is 3.13 × 108 to 1.0 × 1010 total particles/mL, and both the intra- and inter-assay variabilities are less than 5%.

Highly Efficient and Selective CAR-Gene Transfer Using CD4- and CD8-Targeted Lentiviral Vectors

Chimeric antigen receptor (CAR)-modified T cells have revealed promising results in the treatment of cancer, but they still need to overcome various hurdles, including a complicated manufacturing process. Receptor-targeted lentiviral vectors (LVs) delivering genes selectively to T cell subtypes may facilitate and improve CAR T cell generation, but so far they have resulted in lower gene delivery rates than conventional LVs (vesicular stomatitis virus [VSV]-LV). To overcome this limitation, we studied the effect of the transduction enhancer Vectofusin-1 on gene delivery to human T cells with CD4- and CD8-targeted LVs, respectively, encoding a second-generation CD19-CAR in conjunction with a truncated version of the low-affinity nerve growth factor receptor (ΔLNGFR) as reporter. Vectofusin-1 significantly enhanced the gene delivery of CD4- and CD8-LVs without a loss in target cell selectivity and killing capability of the generated CAR T cells. Notably, delivery rates mediated by VSV-LV were substantially reduced by Vectofusin-1. Interestingly, a transient off-target signal in samples treated with Vectofusin-1 was observed early after transduction. However, this effect was not caused by uptake and expression of the transgene in off-target cells, but rather it resulted from cell-bound LV particles having ΔLNGFR incorporated into their surface. The data demonstrate that gene transfer rates in the range of those mediated by VSV-LVs can be achieved with receptor-targeted LVs.

Production of Influenza C HEF pseudotyped lentiviral particles

While influenza A and B virus contain the two glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) inserted into the viral membrane, influenza C virus possesses only one spike designated Hemagglutinin-Esterase-Fusion (HEF) protein which combines the functions of both HA and NA. Like HA, it recognizes and binds to a receptor on the cell surface to initiate virus entry. However, the receptor is N-acetyl-9-O-acetylneuraminic acid. HEF is the receptor-destroying enzyme, which is the function of the neuraminidase (NA) in influenza A and B virus. Although Influenza C virus infections are generally mild and self-limited, it comprises around 13 % of influenza positive cases in hospitalized children. It is also a cause of community acquired pneumonia of children. As influenza research has been more focused on Influenza A and B, it is of interest to pseudotype influenza C HEF. The full sequence of Inflenza C HEF (C/Tokyo/4/2014) was obtained and cloned into pi.18 expression plasmid. The second generation packaging construct pCMVΔR8.91, and the self-inactivating lentiviral vector pCSFLW expressing firefly luciferase, and pCSGW expressing GFP were used for lentiviral vector particle production. The original HEF sequence was mutated by site directed mutagenesis to inhibit esterase activity to prevent receptor destruction prior to pseudotype viral entry. Pseudotyped particles bearing the HEF glycoprotein were successfully produced and verified by fluorescence and Luciferase titration assay with titres of 5×107 RLU/ml. Currently, optimization is undergoing to achieve higher titres as well determining the specific protease/s utilized by HEF for its activation.

Robust Enhancement of Lentivirus Production by Promoter Activation

Lentiviral vectors are a valuable tool to deliver exogenous genes for stable expression in cells. While much progress has been made in processing lentiviral vector-containing culture medium, it remains to be explored how the production of lentiviral vector from producer cells can be increased. We initially found that co-expression of the SPRY domain-containing SOCS box protein 1 (SPSB1) promotes the production of human immunodeficiency virus type 1 (HIV-1) and lentiviral vector with increased expression of the Gag and envelope proteins and activation of the HIV-1 LTR and CMV promoter. The presence of AP-1, NF-κB and CREB/ATF recognition sites in these promoters prompted us to utilize human T-lymphotropic virus type 1 (HTLV-1) Tax for lentiviral vector production because Tax activates all these transcription factors. Co-expression of a small amount of Tax markedly increased both the expression of viral structural proteins in producer cells and release of lentiviral vector particles, resulting in a more than 10-fold enhancement of transduction efficiency. Of note, the Tax protein was not detected in the lentiviral vector particles concentrated by ultracentrifugation, supporting the safety of this preparation. Collectively, these results indicate that promoter activation in producer cells represents a promising approach to preparing high-titer lentiviral vectors.

MicroRNA Profile in Swimming Trained Female Rats and its Therapeutic Potential in Spontaneously Hypertensive Rats

IntroductionBoth aerobic exercise training (ET) and Hypertension (HY) induce different cardiac hypertrophy (CH) phenotypes which molecular differences and may lead to new targets for therapies in cardiovascular disease, as microRNAs (miRNAs).MethodsWe selected 15 miRNAs that were differentially expressed by ET from miRNAarray data and compared them with other from SHR (Spontaneously Hypertensive Rats) miRNAarray data. Four miRNAs were selected for qRT‐PCR confirmation: miRNA‐27a, 27b, 126 e 29c. Among then, miRNA 29c was chosen to be modulated by lentiviral vector due its role in fibrosis regulation. Intramuscular cardiac injection of the lentiviral vector particles 0.6×109 vp/was performed for two different times (7 and 14 days) (SHR7 and SHR14). The transduction efficiency was assessed by GFP expression by Western Blot. Blood pressure (BP) was measured by caudal plethysmography, CH was analyzed by ratio LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) and by cardiomyocytes diameter (dCMO). qRT‐PCR was used to assess miRNA‐29c expression and its targets COLIAI and COLIIIAI gene expression. The LV collagen content was assessed by histology (Picrossirius red), by collagen volume fraction, and by hydroxiproline concentration.ResultsBoth groups that received the low doses were GFP positive in the heart and liver tissue. BP was increased to SHR7, but it was prevented by miR‐29c lentivirus treatment to SHR14. The miRNA‐29c expression increased in all treated groups versus their control (CSI). COLIAI expression decreased in treated groups, while COLIIIAI did not change. Collagen volume fraction decreased in all treated groups, which shows that the treatment was efficient to decrease the cardiac collagen. HW/BW ratio decreased 7–11% in SHR14, while the dCMO was increased in all treated groups.ConclusionsThese results suggest that miR‐29c lentivirus treatment induces beneficial cardiac remodeling in SHR, increasing dCMO and decreasing cardiac fibrosis. These results open perspective for further investigation of antifibrotic therapies for cardiovascular diseases.Support or Funding InformationFAPESP, CNPq, CAPES and NAPmirThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Display of the Albumin-Binding Domain in the Envelope Improves Lentiviral Vector Bioavailability.

Vesicular stomatitis virus G glycoprotein (VSVg) is extensively used for retroviral and lentiviral vector (LV) pseudotyping. However, VSVg pseudotyped vectors are serum inactivated, blocking the in vivo gene delivery. Several strategies have been employed to prevent complement inactivation, including chemical and genetic envelope modifications. This study employed the streptococcal albumin-binding domain (ABD) to generate a construct to express ABD as a glycosylphosphatidylinositol-anchored protein. LV particles bearing ABD are able to bind bovine and human serum albumin in vitro. Neither the lentiviral vector production titer nor the in vitro transduction was affected by the ABD display. The study demonstrated that ABD-bearing LVs are protected from human complement inactivation. More importantly, intravenous administration demonstrated that the presence of ABD significantly reduces lentivector sequestration in liver and bone-marrow cells. Therefore, the use of ABD represents an improvement for in vivo gene therapy applications. The results strongly point to ABD display as a universal strategy to increase the in vivo efficacy of different viral vectors.

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

(RT)-qPCR for detection of and differentiation between RNA and DNA of HIV-1-based lentiviral vectors

The purpose of the described method is the detection of and differentiation between RNA and DNA of HIV-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering operations methods for the detection of the HIV-1 based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here a qPCR method targeting the LTR U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.