Gene Transfer Methods Research Articles

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

Abstract B65: CT-0508, a novel CAR macrophage product directed against HER2, promotes a proinflammatory tumor microenvironment

Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are usually the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor-associated macrophages (TAMs), they participate in disease progression. The current goals of macrophage-based immunotherapies are to reduce TAM infiltration or enhance TAM phagocytosis. In contrast, we have developed a new paradigm based on the adoptive transfer of genetically engineered chimeric antigen receptor (CAR) macrophages (CAR-M) for the treatment of human cancer. CAR-M can only be produced using a unique adenoviral vector, since human macrophages are highly resistant to other methods of gene transfer. We have previously shown that the primary mechanism of action of CAR-M is phagocytosis, and that a single dose of primary human anti-HER2 CAR-M led to significantly improved overall survival in multiple xenograft models. We now establish that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique proinflammatory and antitumor M1 phenotype. Functional evaluation and RNA sequencing revealed that CT-0508 maintain a proinflammatory M1 phenotype despite challenge with immunosuppressive environments in vitro, highlighting their resistance to subversion. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells, we established a novel xenografted human TME model. We demonstrate with single-cell resolution that CT-0508 maintain their phenotype within the human TME. Additionally, CT-0508 activated the human TME and generated an activated human dendritic cell signature. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with immunosuppressive macrophages, dendritic cells, and T cells. CT-0508 shifted bystander macrophages toward a proinflammatory phenotype, induced activation and maturation markers on DCs, and recruited resting as well as activated T cells in chemotaxis assays. Lastly, CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages. These results show that in addition to direct antitumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of activating the solid cancer TME and promoting a proinflammatory phenotype. The safety of CT-0508 will be evaluated in an upcoming first-in-human phase I clinical trial. Citation Format: Konrad Gabrusiewicz, Nicholas Anderson, Xueqing Lu, Xinhe Shan, Olga Shestova, Nicholas Petty, Feng Shen, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Katherine Cummins, Saar Gill, Michael Klichinsky. CT-0508, a novel CAR macrophage product directed against HER2, promotes a proinflammatory tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B65.

Tissue suction-mediated gene transfer to the beating heart in mice.

We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that −75 kPa and −90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at −75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.

Transformation of fluoroquinolone-resistance Neisseria gonorrhoeae gyrA and parC genes

Introduction: N. gonorrhoeae is one of the etiological causative agents of one of the most common sexually transmitted diseases. Gonococci has created many resistance mechanisms, which is associated with bacterial evolution. Natural transformation is the basic method of horizontal gene transfer in bacteria of the genus Neisseria, which can lead to a mutation in the gyrA gene encoding DNA gyrase. The aim of the study was to verify the view on the significance of mutations at positions 91 and 95 of the gyrA protein on the sensitivity of N. gonorrhoeae to antibiotics of the quinolone type. Methods: GyrA gene was introduced into an sensitive isolate of N. gonorrhoeae using genetic transformation. Resistance gene donor, recipient and transform strains were tested for susceptibility and the gyrA gene was sequenced. Results: It has been shown that double mutation in amino acid acid sequence of the GyrA protein at positions 91 and 95 increase the value of MIC from 0,003 mg / L to 0,125 mg / L at CIP sensitive N. gonorrhoeae strain. Conclusions: Mutations in the amino acid sequence at positions 91 and 95 affet the strain’s sensitivity to ciprofloxacin, but it is not the only mechanism which could alter the MIC value of quinolones.

Genetic Transformation Methods for Crop Improvement- A Brief Review

Decades of documented successful pieces of evidence in agritech have shown us a clear picture of the importance of biotechnology in crop improvement. The production in agriculture should have steady growth and to achieve these objective conventional methods should go on parallel with biotechnological approaches. Genetic engineering which has revolutionized the path of crop improvement involves the identification and transfer of novel genes into the existing elite cultivars. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect method of gene transfer has its own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy, elite and eco-friendly method to transfer genes. The transformation method which is a base of genetic engineering is vital and Agrobacterium-mediated gene transfer and gene gun have shown to be doing well in recent years. As a whole the methodology involved, merits and demerits of different methods have been briefly discussed.

Evaluation of vector mediated gene therapy as a novel approach to mitigate cardiovascular diseases; A short review

Positioning in the top list of mortality and morbidity, cardiovascular disease (CVD) is required new approaches to mitigate the risk and prevalence. Recombinant DNA (rDNA) technology and genome sequencing explore the new horizon to treat CVD through modified therapy. Gene transfer mechanism eventually angiogenic gene therapy provides efficacy in early-stage heart failure. Targeted gene delivery system depicts the novel therapeutic pattern for both genetic disorder and pathophysiological deficit and opens the pathway of early diagnose and treatment. Despite some limitations of a specific vector (viral vector) mediated gene transfer, these new approaches make hope on CVD treatment rather than the surgery and other ways. Furthermore, some vectors are less hazardous than viral vectors to suggest successful non-viral gene modulations to Cardiovascular tissues and perform in the clinical trial. However, plasmid-mediated gene transfer method along with genome editing technology might be the best possible approach for future CVD prognostics and diagnostics.

Diversity in Natural Transformation Frequencies and Regulation across Vibrio Species.

In Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in Vibrio campbellii strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4 or Vibrio parahaemolyticus in contrast to the established regulatory pathway in Vibrio cholerae in which quorum sensing is required to activate the competence regulator QstR. Similar to V. cholerae, expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related Vibrio strains, with V. vulnificus having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus To explore differences in competence regulation, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the Vibrio genus.IMPORTANCE Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus.

Gene transfer to German chamomile (L chamomilla M) using cationic carbon nanotubes

The use of nanoparticles to transfer genes to plant cells can solve some problems encountered in other gene transfer methods, including limited host range in the use of Agrobacterium, cell wall removal in the use of polyethylene glycol and electroporation, and high cell damage while using a gene gun. In this research, cationic carbon nanotubes (CNTs) were used to transfer ssDNA-FITC to German chamomile cells. The ability of nanoparticles to interact with and protect of DNA against enzymes and ultrasound damages was investigated using 0.8 % agarose gel. To investigate the morphology of CNTs loaded with DNA (Nanotube- Polyethyleneimine/DNA nanoparticles), scanning electron microscopy (SEM) was used. The biocompatibility effect of CNTs (Nanotube- Polyethyleneimine) on German chamomile cells was also determined by trypan blue staining. Agarose gel images showed that CNTs have a high ability to interact with DNA and can effectively protect it from damage by ultrasound and digestive enzymes. In addition, the SEM images of CNTs/DNA nanoparticles showed that these nanoparticles were rod-shaped with lengths around 100–200 nm. The fluorescence microscope results from German chamomile cells treated with CTNs/ssDNA-FITC nanoparticles showed the ability of these nanoparticles to transfer ssDNA-FITC to German chamomile cells. The results also revealed that the simultaneous use of ultrasound and CNTs significantly increased the transfer efficiency of ssDNA-FITC.

Preclinical Assessment of Non-Virally Engineered CD33.CAR Cytokine-Induced Killer (CIK) Cells in Chemoresistant AML Patient-Derived Xenografts

Background Chimeric Antigen Receptor (CAR)-T cell therapy has been successfully clinically deployed in the context of B-cell malignancies, paving the way for further development also in Acute Myeloid Leukemia (AML), a still unmet clinical need in the field of oncohematology. Among the potential AML targetable antigens, CD33 is so far one of the main validated molecule. Objectives The aim of the present study was to optimize a non-viral gene transfer method to engineer Cytokine-Induced Killer (CIK) cells with a CD33.CAR by using a novel version of the Sleeping Beauty (SB) transposon system, named "SB100X-pT4". Further, a preclinical assessment of SB-modified CD33.CAR-CIK cells was performed in chemoresistant AML Patient-Derived Xenografts (PDX), in order to address the unmet need of targeting drug-resistant AML cells. Methods Donor derived-CIK cells were stably transduced with a CD33.CAR by exploiting the novel hyperactive SB100X transposase and the pT4 transposon (SB100X-pT4). The novel SB system has been in vitro compared to the previous established SB11-pT. In vitro anti-AML activity of CD33.CAR-CIK cells was assessed by flow cytometry-based cytotoxicity (AnnV-7AAD), proliferation (CFSE) and cytokine production (intracellular IFNg and IL2 detection) assays. In vivo efficacy was evaluated in NSG mice transplanted with MA9-NRas AML cell line or PDX samples. A xenograft chemotherapy model mimicking induction therapy ("5+3" Ara-C and doxorubicin) was exploited to examine the potential benefit of CD33.CAR-CIK cells on chemoresistant/residual AML cells. Results By significantly reducing the amount of DNA transposase, the novel SB100X-pT4 combination resulted in higher CAR levels than the SB11-pT. SB100X-pT4-modified CD33.CAR CIK cells showed efficient expansion after 3 weeks (median fold increase of 38.89, n=4). Both transpositions conferred to CD33.CAR-CIK cells a specific killing (up to 70%) against CD33+ AML target cell lines and primary AML cells. The anti-AML proliferative response of SB-modified CD33.CAR-CIK cells was also considerable (up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% for IFN-γ and up to 25% for IL-2). To evaluate the effect of SB100X-pT4-modified CD33.CAR-CIK cells particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an "early treatment" in mice transplanted with the MA9-NRas cell line, which retains a high frequency of LICs. At sacrifice, CD33.CAR-CIK cell-treated mice showed a significant bone marrow (BM) engraftment reduction (median 27.80 for the untreated group and 22.60 for the unmanipulated CIK cells vs 6.45 for CD33.CAR-CIK cell, n=4 NSG mice per group, p= 0.02). PDX of two different AML samples at the onset were established to be used as models mimicking different disease conditions. In an "early treatment" model using secondary transplanted PDX, a setting which presumably reflects the typical LIC properties, a clear engraftment reduction in the treated cohort was observed, nearly undetectable in 2/5 mice, as compared to the untreated mice (up to 70% in BM). A significant leukemia reduction was also measured in the peripheral blood and spleen of treated mice, showing CD33.CAR-CIK cell potential of reducing AML dissemination in the periphery. When ex vivo re-exposed to CD33.CAR-CIK cells residual AML cells were still sensitive to the treatment, indicating that no resistance mechanisms occurred. CD33.CAR-CIK cells were also effective in a second model by which the treatment started when AML engraftment was clearly manifested in the BM (> 1%). Finally, when starting CD33.CAR-CIK cell treatment after disease recurrence post induction therapy, a significant disease reduction was observed in the CD33.CAR-CIK-treated group, reaching undetectable levels in half of the mice, as compared to chemotherapy-only treated mice (up to 60% of engraftment in BM)(Figure 1). Conclusions The employment of a non-viral SB-based CD33.CAR-gene transfer approach, which is overall associated to less cumbersome protocols and reduces the cost of goods, offers a unique alternative to current viral-based strategies to be explored in the setting of resistant forms of AML. Disclosures No relevant conflicts of interest to declare.

Production of Lentivirus for the Establishment of CAR-T Cells.

One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.

Highly efficient ex vivo lentiviral transduction of primary human pancreatic exocrine cells

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. Therefore, improved gene transfer protocols are needed. Lentiviral vectors are widely used to drive ectopic gene expression in mammalian cells, including primary human islet cells. Here we aimed to optimize gene transfer into primary human exocrine cells using modified lentiviral vectors or transduction conditions. We evaluated different promoters, viral envelopes, medium composition and transduction adjuvants. Transduction efficiency of a reporter vector was evaluated by fluorescence microscopy and flow cytometry. We show that protamine sulfate-assisted transduction of a VSV-G-pseudotyped vector expressing eGFP under the control of a CMV promoter in a serum-free environment resulted in the best transduction efficiency of exocrine cells, reaching up to 90% of GFP-positive cells 5 days after transduction. Our findings will enable further studies on pancreas (patho)physiology that require gene transfer such as gene overexpression, gene knockdown or lineage tracing studies.

Sperm-Mediated Genetic Modifications.

The ability to introduce controlled modifications of the genome of animals represents an important tool for biomedical and veterinary research. Among transgenic techniques, we describe here the sperm-mediated gene transfer method that is based on the spontaneous ability of sperm cells to bind and internalize exogenous DNA and to carry it to theoocyte during fertilization, producing genetically modified animals.

Optimization and Characterization of Calcium Phosphate Transfection in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a promising tool for cell therapy, and gene-modified MSCs further expand their applications. To take full advantage of MSCs as a therapeutic approach, developing effective gene transfer methods is critical. Calcium phosphate transfection is well-established and safe, but the protocols need to be optimized according to different cell types. Currently, there is no optimized protocol for MSCs. This study optimized the protocol of calcium phosphate transfection for MSCs and highlighted the importance of serum during the process of transfection. More interestingly, the behavior of gene overexpression in MSCs in the in vivo environment was verified.

A Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy.

Genetic engineering is an important tool for redirecting the function of various types of immune cells and their use for therapeutic purpose. Although NK cells have many beneficial therapeutic features, genetic engineering of immune cells for targeted therapy focuses mostly on T cells. One of the major obstacles for NK cell immunotherapy is the lack of an efficient method for gene transfer. Lentiviral vectors have been proven to be a safe tool for genetic engineering, however lentiviral transduction is inefficient for NK cells. We show in this study that lentiviral vectors pseudotyped with a modified baboon envelope glycoprotein can transduce NK cells 20-fold or higher in comparison to VSV-G pseudotyped lentiviral vector. When we investigated the mechanism of transduction, we found that activated NK cells expressed baboon envelope receptor ASCT-2. Further analysis revealed that only a subset of NK cells could be expanded and transduced with an expression profile of NK56bright, CD16dim, TRAILhigh, and CX3CR1neg. Using CD19-CAR, we could show that CD19 redirected NK cells efficiently and specifically kill cell lines expressing CD19. Taken together, the results from this study will be important for future genetic modification and for redirecting of NK cell function for therapeutic purpose.

Osteopetrotic induced pluripotent stem cells derived from patients with different disease-associated mutations by non-integrating reprogramming methods

BackgroundAutosomal recessive osteopetrosis is a genetically and phenotypically heterogeneous disease, caused by defects in osteoclast formation and function. The only available treatment is allogeneic stem cell transplantation that has still high morbidity and mortality. The goal of the present study was to generate iPSCs from bone marrow-derived MSCs of osteopetrosis patients with three most common mutations by using two different integration-free gene transfer methods and compare their efficiencies. The secondary objective was to select the most appropriate integration-free production method for our institutional iPSC bank using this rare disease as a prototype.MethodsTwo different integration-free gene transfer methods (episomal and Sendai viral vectors) were tested and compared on the same set of patient samples exhibiting three different mutations associated with osteopetrosis. Generated iPSCs were characterized by standard assays, including immunophenotyping, immunocytochemistry, RT-PCR, embryoid body, and teratoma assays. Karyotype analyses were performed to evaluate genetic stability.ResultsiPSC lines exhibiting typical ESC-like colony morphology were shown to express pluripotency markers by immunofluorescence staining. Over 90% of the cells were found positive for SSEA-4 and OCT3/4 and negative/weak positive for CD29 by flow cytometry. Immunohistochemical staining of teratoma and spontaneously differentiated embryoid body sections confirmed their trilineage differentiation potential. All iPSC lines expressed pluripotency-related genes. Karyotype analyses were found normal. Direct sequencing of PCR-amplified DNA showed that disease-related mutations were retained in the patient-specific iPSCs.ConclusionGeneration of iPSC using SeV and episomal DNA vectors have several advantages over other methods like the ease of production, reliability, high efficiency, and safety, which is required for translational research. Furthermore, owing to the pluripotency and self-renewal capacity, patient-specific iPSCs seem to be ideal cell source for the modeling of a rare genetic bone disease like osteopetrosis to identify osteoclast defects, leading to clinical heterogeneity in osteopetrosis patients, especially among those with different mutations in the same gene.

Abstract 4772: Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer

Abstract Human Adenoviruses (hAd) have been broadly used as gene delivery tool in preclinical and clinical studies of cancer gene therapy. The main challenges associated with systemic delivery of hAds are their high immunogenicity and host-specificity. Adenoviruses can elicit strong innate and acquired immune responses that reduce therapeutic gene transfer and expression in malignant sites. Murine cells generally lack some of the receptors necessary for hAd adhesion and internalization making them not permissive to infection and replication and limiting translational studies using murine models. We have developed a gene transfer method, which uses lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to protect the hAds from the immune system and to deliver them to a targeted tissue, bypassing the necessity for specific receptors. The expression of the Ad-receptors (CAR, Integrins αvβ3 and αvβ5), transduction efficiency of Ad-GFP, and GFP transgene expression were measured in vitro in murine (TRAMP-C2) and human (DU145) prostate cancer cells lines by flow cytometry and immunofluorescence. The activation of the innate and acquired immune system following the injection of Ad-GFP/MBs complexes was evaluated in vivo in C57BL/6 mice healthy or bearing a TRAMP-C2 syngeneic tumor graft. ELISA was used to quantify the inflammatory cytokines TNF-α, IL-6 and measure Neutralizing Antibodies. ELISpot assay was performed to measure the frequency of DBP-specific CD8+ T cells secreting INF-γ. We showed in vitro that murine TRAMP-C2 cells display an expression pattern of Ad-receptors comparable to human DU145 prostate cancer cells. We also demonstrated that both murine and human cells showed a dose dependent increase in the percentage of cells transduced by Ad-GFP at 24 hours. Additionally, we showed that TRAMP-C2 cells efficiently express the GFP transgene at 48 and 72 hours post transduction. To assess in vivo if our gene transfer method could effectively protect the Ads form both the innate and adaptive immunity, we injected C57BL/6 mice with the hAds-GFP/MBs complexes +/-US. Notably we did not observe activation of either innate (secretion of TNF- and IL-6 cytokines) or acquired immunity (neutralizing antibodies and presence of adenovirus-specific CD8+ T cells producing INF-γ). Our data provides evidence that the TRAMP-C2 syngeneic model of prostate cancer is a suitable system to study in immunocompetent mice the capability of the MBs/US to protect the Adenoviruses from the immune system while delivering them to a targeted site bypassing the requirement of specific receptors. Citation Format: Flavia De Carlo, Elliot T. Varney, Pier Paolo Claudio, Candace M. Howard. Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4772.

The use of transgenic mosquitoes for prevention of spread of arboviral diseases.

The mosquitoes of Aedes genus are the most important vector such arboviral diseases as dengue, yellow, Chikungunya, West Nile and Zika fevers. Work is currently in progress to control the transmission of agents of these diseases by forming of transgenic mosquitoes in order to altering the capacity of wild mosquitoes to support of virus replication. There are two main strategies of genetic control of mosquitoes population. Sterile Insect Technique (SIT), that mainly uses population suppression methods for making self-sustaining genetic systems and Release of insects carrying of a Dominant Lethal (RIDL) that uses mainly gene transfer methods for making of self-limiting genetic systems. The RIDL is more expensive, but it has some significant preferences, according compares with SIT. The field trials of genetic control methods are conducted in several countries from 2009 to present time. Genetic control, transgenic technologies to induce sterility, genetic elimination and stable transformation of Aedes mosquitoes are viewed in this review.

PD-L1 Ameliorates Murine Acute Graft-Versus-Host Disease by Suppressing Effector But Not Regulatory T Cells Function.

There is increasing evidence that interaction between programmed death 1 (PD-1) and its ligands PD-1 (PD-L1) plays a critical role in the pathology of acute graft-versus-host disease (aGVHD). However, the role of PD-L1 in the development of aGVHD has been controversial in recent mouse studies. In this study, we carried out studies in a murine aGVHD model to clarify the role of PD-L1 in aGVHD pathogenesis. We found that systemic overexpression of PD-L1 by hydrodynamic gene transfer (HGT) method in vivo ameliorates aGVHD-induced lethality in mice. Systemic overexpression of PD-L1 inhibits the donor T cells activation, effector memory status, as well as Th1 and Th17 cells responses in vivo. In addition, PD-L1 Ig treatment significantly suppressed T cells' proliferation, promoted T cells' apoptosis, and reduced pro-inflammatory cytokines expression by effector T cells in vitro in the stimulation of anti-CD3/CD28 and allogeneic dendritic cells. However, we found that PD-L1 overexpression did not affect Treg cells' differentiation in vivo and in vitro, depletion of Treg cells in PD-L1 HGT recipients did not aggravate aGVHD mortality. Therefore, our results demonstrated that systemic treatment with PD-L1 protein ameliorates aGVHD by suppressing effector but not regulatory T cell function. Our findings suggest that systemic treatment with PD-L1 may be a potential strategy to prevent or ameliorate aGVHD.

Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method

Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.Keywords: cloning, epigenetics, lipofection, lentiviral transduction, nuclear reprogramming. Resumen Antecedentes: La producción de animalestransgénicossigue siendo una biotecnología de baja eficiencia, y se deberían utilizar alternativas sencillas para mejorar la tasa de producción de bovinos transgénicos mediante transferencia nuclear. Una de estas alternativas es la selección del tipo mas apropiado de célula donante y método de transferencia génica. Objetivo: Investigar el efecto del tipo celular (fibroblastos fetales o adultos, y celulas del cumulus), y el método de transferencia génica (lipofección y transducción lentiviral) en la incorporación, expresión génica, y la intensidad de fluorescencia del transgén en células bovinas analizadas por citometría de flujo. Métodos: Fibroblastos fetales (FF), fibroblastos adultos (AF), y células del cúmulo (CC) fueron transfectados a través de lipofección o transducidos utilizando partículas lentivirales producidas con plásmidos que expresan la proteína verde fluorescente (GFP). Resultados: La transducción lentiviral dio lugar a mayores tasas de expresión del transgen en todos los tipos de células (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96, CC: 60,7% ± 14,7) en comparación con la lipofección (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). Las células del cúmulus mostraron menores tasas de expresión del transgen que los otros tipos celulares. En cuanto a la intensidad de fluorescencia, no hubo diferencias significativas entre lipofección y transducción lentiviral; en ambos tratamientos, se obtuvo una mayor intensidad de fluorescencia cuando se usaron células adultas en lugar de células fetales. Conclusión: La eficiencia de la transferencia de genes varía según el tipo de célula y el método de transferencia génica, con la transducción lentiviral se logra una mayor tasa de transfección, y los fibroblastos adultos muestran una mejor expresión transgénica.Palabras clave: clonación, epigenética, lipofección, reprogramación nuclear, transducción lentiviral. Resumo Antecedentes: A produção de animais transgênicos é uma biotecnologia que ainda apresenta baixa eficiência e alternativas simples devem ser usadas para o aumento da produção de bovinos transgênicos por transferência nuclear. Uma destas alternativas compreende a seleção do tipo apropriado de célula doadora de núcleo e do método de transferência gênica. Objetivo: Investigar a influência do tipo celular (fibroblastos fetais ou adultos, e células do cumulus), e do método de transferência gênica (transfecção por lipofecção ou transdução lentiviral) na incorporação, expressão, e na intensidade de fluorescência do transgene em células bovinas analisadas por citometria de fluxo. Métodos: Fibroblastos fetais (FF), fibroblastos adultos (AF), e células do cumulus (CC) foram submetidas à lipofecção ou à transfecção lentiviral utilizando plasmídeos expressando a Proteína Fluorescente Verde – GFP). Resultados: A transdução lentiviral resultou em maiores taxas de expressão do transgene em todos os tipos celulares (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96; CC: 60,7% ± 14.7) quando comparada com a lipofeccção (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). As células do cumulus apresentaram menores taxas de expressão quando comparadas aos outros tipos celulares. Com relação à intensidade de fluorescência, não houve diferença significativa entre a lipofecção e a transdução lentiviral e em ambos os tratamentos as células adultas apresentaram maior intensidade de fluorescência do que as células fetais. Conclusão: A eficiência de transferência gênica varia de acordo com o tipo celular, e com o método de transferência gênica, sendo que a transdução lentiviral resultou em maiores taxas, e que os fibroblastos adultos mostraram melhor expressão do transgene.Palavras-chave: clonagem, epigenética, lipofecção, reprogramação nuclear, transdução lentiviral.

A centrifugation-assisted peptide-mediated gene transfer method for high-throughput analyses.

A peptide-mediated DNA delivery system for several plant species has been recently developed. This system uses ionic complexes of DNA and fusion peptides containing several domains, such as DNA-binding and cell-penetrating peptides. Although the peptide-DNA complexes are capable of penetrating into plant cells through the cell wall by mechanical pressure using a syringe, sample throughput is limited. Here, we describe a Centrifugation-Assisted Peptide-mediated gene Transfer (CAPT) method for improving sample throughput with reproducible gene transfer efficiency. We optimized the parameters of CAPT for transient gene transfer efficiency by using Nicotiana tabacum cotyledons as a model plant material. The optimal parameters for centrifugation were 10,000×g for 60 s. Furthermore, we successfully transferred the peptide-DNA complex into rice cotyledons using the optimized CAPT method. Thus, the CAPT method is superior to the previous syringe-mediated infiltration method in terms of sample throughput in multiple plant species.