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The coronavirus disease (COVID-19) pandemic has increased pressure to develop low-cost, compact, user-friendly, and ubiquitous virus sensors for monitoring infection outbreaks in communities and preventing economic damage resulting from city lockdowns. As proof of concept, we developed a wearable paper-based virus sensor based on a molecular imprinting technique, using a conductive polyaniline (PANI) polymer to detect the lentivirus as a test sample. This sensor detected the lentivirus with a 4181 TU/mL detection limit in liquid and 0.33% to 2.90% detection efficiency in aerosols at distances ranging from 30 cm to 60 cm. For fabrication, a mixture of a PANI monomer solution and virus were polymerized together to form a conductive PANI sensing element on a polyethylene terephthalate (PET) paper substrate. The sensing element exhibited formation of virus recognition sites after the removal of the virus via ultrasound sonication. A dry measurement technique was established that showed aerosol virus detection by the molecularly imprinted sensors within 1.5 h of virus spraying. This was based on the mechanism via which dispensing virus droplets on the PANI sensing element induced hybridization of the virus and molecularly imprinted virus recognition templates in PANI, influencing the conductivity of the PANI film upon drying. Interestingly, the paper-based virus sensor was easily integrated with a wearable face mask for the detection of viruses in aerosols. Since the paper sensor with molecular imprinting of virus recognition sites showed excellent stability in dry conditions for long periods of time, unlike biological reagents, this wearable biosensor will offer an alternative approach to monitoring virus infections in communities. [ABSTRACT FROM AUTHOR]

The discovery of HIV-1 as the cause of AIDS was one of the major scientific achievements during the last century. Here the events leading to this discovery are reviewed with particular attention to priority and actual contributions by those involved. Since I would argue that discovering HIV was dependent on the previous discovery of the first human retrovirus HTLV-I, the history of this discovery is also re-examined. The first human retroviruses (HTLV-I) was first reported by Robert C. Gallo and coworkers in 1980 and reconfirmed by Yorio Hinuma and coworkers in 1981. These discoveries were in turn dependent on the previous discovery by Gallo and coworkers in 1976 of interleukin 2 or T-cell growth factor as it was called then. HTLV-II was described by Gallo's group in 1982. A human retrovirus distinct from HTLV-I and HTLV-II in that it was shown to have the morphology of a lentivirus was in my mind described for the first time by Luc Montagnier in an oral presentation at Cold Spring Harbor in September of 1983. This virus was isolated from a patient with lymphadenopathy using the protocol previously described for HTLV by Gallo. The first peer reviewed paper by Montagnier's group of such a retrovirus, isolated from two siblings of whom one with AIDS, appeared in Lancet in April of 1984. However, the proof that a new human retrovirus (HIV-1) was the cause of AIDS was first established in four publications by Gallo's group in the May 4th issue of Science in 1984. [ABSTRACT FROM AUTHOR]

Microglia are resident immune cells in the central nervous system (CNS) that play vital roles in CNS development, homeostasis and disease pathogenesis. Genetic defects in microglia lead to microglial dysfunction, which in turn leads to neurological disorders. The correction of the specific genetic defects in microglia in these disorders can lead to therapeutic effects. Traditional genetic defect correction approaches are dependent on viral vectorbased genetic defect corrections. However, the viruses used in these approaches, including adeno-associated viruses, lentiviruses and retroviruses, do not primarily target microglia; therefore, viral vector-based genetic defect corrections are ineffective in microglia. Microglia replacement is a novel approach to correct microglial genetic defects via replacing microglia of genetic defects with allogenic healthy microglia. In this paper, we systematically review the history, rationale and therapeutic perspectives of microglia replacement, which would be a novel strategy for treating CNS disorders. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer. [ABSTRACT FROM AUTHOR]

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains. [ABSTRACT FROM AUTHOR]

Laboratory wastewater has been suggested as an important escape route for microorganisms from research environments. Likely reasons for the unintentional release of laboratory organisms are shortcomings in the handling of infectious liquid biological waste (LBW) and inadequate inactivation procedures. We developed an analytical approach to investigate the use of chemical inactivation (CI) procedures in Swiss research laboratories by on-site random sampling of presumably inactivated infectious LBW and testing it for the presence of infectious lentiviruses (HIV-1) and adenoviruses (AdV). In addition, standard operating procedures (SOPs) for CI were collected and evaluated, and laboratorystaff knowledge of CI processes was assessed using a questionnaire. Although we found several deficiencies in the technical knowledge and training of laboratory staff on the CI of LBW, as documented by 27 returned questionnaires, no infectious viruses were detected in the eight LBW samples collected. Whilst we acknowledge that the number of LBW samples and SOPs is small, we conclude that CI of LBW containing infectious lentiviruses and adenoviruses does not result in the systematic release of considerable amounts of infectious viruses into the environment from research laboratories in Switzerland. [ABSTRACT FROM AUTHOR]

Simple Summary: The characterization of small ruminant lentiviruses in autochthonous goat and sheep populations, like the Moroccan one, represents an excellent source of information to understand their evolution from domestication to present day and improve the knowledge of the genetic diversity of these viruses. Serological data confirmed a low prevalence likely due to extensive breeding and management practice in this area. However, the low rate of success of routinely used serological tests led us to suppose that divergent strains might have escaped diagnostic tools thanks to mutations' diagnostic epitopes. This hypothesis is confirmed by next-generation sequencing results, which highlighted the presence of an A subtype carrying a mismatch in the serotyping epitope. Moreover, the circulation of novel B and recombinant A/B subtypes was also revealed. Recent studies that investigated the origins of SRLV strains offered new insights into their distribution among domestic ruminants. The aim of the study was to investigate SRLV circulation in Morocco. A total of 51 farms were selected in different geographical locations and tested by screening and genotyping ELISA. Whole blood was used for DNA extraction and nested gag PCR. The sample size allowed for an estimation of prevalence lower than 20% (CI 95%). Surprisingly, a large proportion of screening-positive samples were not correctly serotyped. Sanger and NGS amplicon sequencing approaches allowed us to obtain new sequences even from difficult-to-amplify samples. The serological data support the evidence of an intrinsic difficulty of SRLV to spread, likely due to management practices. The low rate of success by genotyping ELISA led us to suppose that divergent strains might have escaped from diagnostic tools, as partially confirmed by the evidence of an A subtype carrying a mismatch in serotyping epitope. The sequence analysis revealed the circulation of novel B and recombinant A/B subtypes. This study highlights the importance of monitoring viral sequences and their evolution to develop specific diagnostic tests, particularly in countries where control measures are in place. [ABSTRACT FROM AUTHOR]

Background: Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies. However, its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity. Localized delivery of tumor-specific T-cells using biomaterials has shown promise, however, procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints. Methods: Polyethylene glycol (PEG)-based three-dimensional (3D) scaffolds were developed and conjugated with positively charged poly-L-lysine (PLL) using carbamide chemistry for efficient loading of lentiviruses (LVs) carrying tumor antigen-specific T-cell receptors (TCRs). The physical and biological properties of the scaffold were extensively characterized. Further, the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin (B16-OVA) tumor model to evaluate the anti-tumor response and the presence of transduced T-cells. Results: Our findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site. In B16-OVA melanoma tumor-bearing mice, the scaffolds efficiently transduce host T-cells with OVA-specific TCRs. These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs, resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines. Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds. Conclusion: Our scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors. This approach offers a viable alternative to in vitro manipulation of T-cells, circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells. It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells, thereby, overcoming a major hurdle. [ABSTRACT FROM AUTHOR]

Lentiviral vectors are valuable tools to express genes of interest in living animals and stem cell cultures. The use of promoters in lentiviral constructs has been successfully used to drive gene expression in particular cell types including neurons and glia of the central nervous system in vivo. However, their suitability in cell culture is less well documented. In this paper, we describe lentiviral vectors containing neuronal promoters of the murine stem cell virus, of the synapsin 1 gene, the tubulin alpha 1 gene, and the calmodulin kinase II gene, and the glial promoter of the glial fibrillary acidic protein gene to drive reporter gene expression in primary dissociated cerebellar cell cultures and in slice cultures. While the glial promoter was highly specific for glia, the neuronal promoters were active in neurons and glia of dissociated cultures to a comparable extent. In slice cultures, neuronal and glial promoters demonstrated higher, but not absolute selectivity for particular cell types. In addition, the promoters allowed for an efficient and graded expression of genes in dissociated cultures. By using selected combinations of vectors, it was also possible to drive the expression of two genes in one cell type with high efficiency. A gene of interest in combination with a reporter gene can thus be expressed in a graded manner to reveal gene function in a rather short time and in a complex cellular environment. [ABSTRACT FROM AUTHOR]

The Human Immunodeficiency Virus - HIV is responsible for causing Acquired Immunodeficiency Syndrome - AIDS. This virus also affects millions of people in the whole world, generating large global impact. It is a member of the lentivirus family of animal retrovirus and has an affinity for defense cells of the body, which the main target is the CD4 + T lymphocytes. Once connected to a component of this cell, the HIV will penetrate it and multiply, destroying them, by weakening the immune system, making it fragile and susceptible to opportunistic diseases. There are two known types of HIV, which are deeply related, named as HIV-1 and HIV-2. HIV-1 is the main cause of AIDS, HIV-2 differs in genomic structure and antigenicity, also causes the disease, but with slower progression. AIDS is the clinical manifestation generated by HIV, once the immune system is compromised. It is estimated that, in Brazil, 718000 people living with HIV / AIDS. There is, currently, no effective vaccine or cure for AIDS, however effective antiretroviral therapies have been used. Through this literature review the paper aimed to show on the HIV virus, its general features, its immunological changes, the national level of this epidemiology, the diagnosis and treatment, warning about the importance of this infectious agent and its prevention, towards control and reduction to the AIDS epidemic. [ABSTRACT FROM AUTHOR]

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Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling. [ABSTRACT FROM AUTHOR]

It is becoming increasingly clear that organisms have developed a variety of mechanisms to fight against viral infection. The viruses have developed means of counteracting these defences in various ways. The APOBEC3 proteins are a mammalian-specific family of nucleic acid cytidine deaminases that block retroviral infection. These inhibitors are counteracted by the Vif proteins encoded by most lentiviruses. In this paper, we will review the interaction of the lentiviral Vif proteins with the APOBEC3 proteins, with an emphasis on sheep APOBEC3 and maedi-visna virus (MVV) Vif. [ABSTRACT FROM AUTHOR]

Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike non-viral methods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome, which can lead to long-term transgene expression. Lentiviruses have advantages over other types of viruses due to their capacity to transduce non-dividing cells. An optimized generation of lentiviruses carrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) or CMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by co-transfecting lentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines. Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine (PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvested at days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFP harvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally, the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transient and stable GFP expression. It was found that the GFP expression declined overtime. However, the transduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/ GFP were higher compared to LV/UbC/GFE In conclusion, we have successfully produced lentiviruses carrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells at different efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to proceed to the subsequent analysis of the effect of regulatory elements in future study. [ABSTRACT FROM AUTHOR]

Abstract: This paper offers an overview of important veterinary viral diseases of mammals stemming from aberrant immune response. Diseases reviewed comprise those due to lentiviruses of equine infectious anaemia, visna/maedi and caprine arthritis encephalitis and feline immunodeficiency. Diseases caused by viruses of feline infectious peritonitis, feline leukaemia, canine distemper and aquatic counterparts, Aleutian disease and malignant catarrhal fever. We also consider prospects of immunoprophylaxis for the diseases and briefly other control measures. It should be realised that the outlook for effective vaccines for many of the diseases is remote. This paper describes the current status of vaccine research and the difficulties encountered during their development. [Copyright &y& Elsevier]

A correction to the article "A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference" published in a 2007 issue of "Nature Genetics" periodical.

Please cite this paper as: Barrett, Parham, Pippal, Cockshell, Moretti, Brice, Pitson, and Bonder (2011). Over-Expression of Sphingosine Kinase-1 Enhances a Progenitor Phenotype in Human Endothelial Cells. Microcirculation 18( 7), 583-597. Abstract Objectives: The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. Methods: The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. Results: Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. Conclusions: These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Stable packaging cell lines have not yet met the growing demand for viral vectors. • Viral plasmid systems have been developed to improve viral titers and safety. • Stable packaging cell lines have been developed to improve viral titers and safety. Lentivirus and other retroviral vectors are useful tools to engineer chimeric antigen receptor (CAR) T cells which are designed to target tumor cells in the growing field of gene therapy. Several efforts have been made to improve vector packaging systems including plasmid systems and envelopes and develop stable packaging cell lines (PCLs) in order to improve the productivity and safety by eliminating cytotoxicity of HIV-1 genes and unnecessary homologous genomic regions. In this paper, we explored stable PCLs developed to date for the production of both lentiviral and retroviral vectors and addressed key features of the viral vector production systems and how the features can be further utilized and modified to meet the growing demand in clinical trials. [ABSTRACT FROM AUTHOR]

Generation of induced pluripotent stem (iPS) cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors (TFs). However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.Cell Research (2009) 19:296–306. doi: 10.1038/cr.2009.20; published online 24 February 2009 [ABSTRACT FROM AUTHOR]

The study of retroviruses has had a profound impact by unveiling an unusual form of viral replication: the multiplication of RNA viruses via a proviral DNA, for which Jan Svoboda provided the experimental model over forty years ago. In 1970 Temin, Mizutani and Baltimore discovered that this group of viruses contains a unique enzyme catalysing the synthesis of a DNA copy of the viral RNA: reverse transcriptase (RT). The discovery of RT has itself had an enormous impact on molecular biology in general, but also stimulated many premature claims of its detection in human disease. Claims by Gallo's laboratory that the cytoplasm of human leukaemia cells contained RT proved to be unfounded, as did his report in collaboration with Weiss that myeloid leukaemia contained HL23 virus, this organism proving not to be human but a laboratory contaminant of three monkey viruses. Conclusive demonstration of a retroviral involvement in human leukaemia was first provided in 1981 by Hinuma and his associates, showing that adult T-cell leukaemia (ATL), a rare form of leukaemia endemic to south-west Japan, is caused by a new retrovirus (ATLV). Other publications in December 1980 and through 1981 claimed the discovery of a new human T-cell leukaemia virus involved in mycosis fungoides (MF) and Sézary's syndrome (SS). This virus was termed HTLV by Gallo. The nucleotide sequence of ATLV is strongly conserved, that of my 1983 isolate from a black British ATL patient being practically identical with the Japanese virus isolates. After AIDS was recognised in 1981 by Gottlieb and coworkers as a new human disease, several papers were published by Gallo and his associates during 1983-4, invoking the oncovirus responsible for adult T-cell leukaemia as the cause of AIDS. In 1983 the French scientist Barré-Sinoussi and her colleagues succeeded in isolating a new agent in the disease, a lentivirus, which they named LAV. The French immunologist Klatzmann and his colleagues discovered... [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLVs) affect sheep and goats worldwide. The major gene related to SRLV infections is the Transmembrane Protein Gene 154 (TMEM154). We estimated the haplotype frequencies of TMEM154 in the USA (USDA-ARS) and Brazil (Embrapa) Gene Banks by using two different SNP genotyping methodologies, FluidigmTM and KASPTM. We also genotyped the ZNF389_ss748775100 deletion variant in Brazilian flocks. A total of 1040 blood samples and 112 semen samples from 15 Brazilian breeds were genotyped with Fluidigm for the SNP ZNF389_ss748775100 and 12 TMEM154 SNPs. A total of 484 blood samples from the Santa Inês breed and 188 semen samples from 14 North American sheep breeds were genotyped with KASP for 6 TMEM154 SNPs. All the Brazilian samples had the "I/I" genotype for the ZNF389_ss748775100 mutation. There were 25 TMEM154 haplotypes distributed across the Brazilian breeds, and 4 haplotypes in the US breeds. Haplotypes associated with susceptibility were present in almost all breeds, which suggests that genetic testing can help to improve herd health and productivity by selecting non-susceptible animals as founders of the next generations. Fluidigm and KASP are reliable assays when compared with Beadchip arrays. Further studies are necessary to understand the unknown role of TMEM154 mutations, host–pathogen interaction and new genes associated with the clinical condition. [ABSTRACT FROM AUTHOR]

Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell-based gene therapies. [ABSTRACT FROM AUTHOR]

During the past decade, successful gene therapies for immunodeficiencies were finally brought to the clinic. This was accomplished through new gene therapy vectors and improved procedures for genetic modification of autologous hematopoietic stem cells. For HIV, autologous hematopoietic stem cell (HSC) gene therapy with ‘anti-HIV genes’ promises a functional cure for the disease. However, to develop such a therapy and translate it into a clinical application is rather challenging. The risks and benefits of such a therapy have to be understood, and regulatory hurdles need to be overcome. In this joint paper by academic researchers and regulators, we are, therefore, outlining a high level roadmap for the early stage development of HSC gene therapy as a potential functional cure for HIV. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLVs) represent a very heterogeneous group of ss-RNA viruses that infect sheep and goats worldwide. They cause important, deleterious effects on animal production and limit the animal trade. SRLVs show a high genetic variability due to high mutation rate and frequent recombination events. Indeed, five genotypes (A–E) and several subtypes have been detected. The aim of this work was to genetically characterize SRLVs circulating in central Italy. On this basis, a phylogenetic study on the gag-pol genetic region of 133 sheep, collected from 19 naturally infected flocks, was conducted. In addition, to evaluate the frequency of mutation and the selective pressure on this region, a WebLogo 3 analysis was performed, and the dN/dS ratio was computed. The results showed that 26 samples out of 133 were clustered in genotype A and 106 samples belonged to genotype B, as follows: A9 (n = 8), A11 (n = 10), A24 (n = 7), B1 (n = 2), B2 (n = 59), and B3 (n = 45). No recombination events were found. Mutations were localized mainly in the VR-2 region, and the dN/dS ratio of 0.028 indicated the existence of purifying selection. Since the genetic diversity of SRLVs could make serological identification difficult, it is important to perform molecular characterization to ensure a more reliable diagnosis, to maintain flock health status, and for the application of local and national control programs. [ABSTRACT FROM AUTHOR]

To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing fulllength GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all Cterminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease. [ABSTRACT FROM AUTHOR]

Abstract: Foxp1 and Foxq1 are two multifunctional molecules of “forkhead box (Fox)” family. The objective of this paper was to construct the lentiviral vectors expressing RNA interference (RNAi) against Foxp1 or Foxq1 genes, and the effects of both vectors with two RNAis on the proliferation, migration and apoptosis of 7721 hepatocarcinoma cell line were evaluated. Six target sequences against human Foxp1/Foxq1 mRNA were designed respectively and six pairs of their corresponding double-strand DNA oligo (siRNA) were synthesized prior to being transfected into 7721 cells with lipo2000, then a most efficient siRNA were selected to be subcloned into pLL3.7-GFP/Lenti plasmids. These plasmids were transfected into 293T cells to package lentiviral particles for subsequent transfection into 7721 cells after their sequences were confirmed. The expression of Foxp1and Foxq1 genes in the transfected cells were identified by real-time PCR. The migration, infiltration, viability and apoptosis of the transfected cells were assessed by wound healing assay, Transwell assay, CCK-8 assay and flow cytometry. Sequencing results showed that lentiviral vectors contained Foxp1 or Foxq1 gene. After being transfected into 7721 cells, Foxp1 and Foxq1 expression were significantly down-regulated by siRNA-823 and siRNA-834. The migration and infiltration ability, and the viability of 7721 cells transfected with two siRNAs were significantly suppressed; flow cytometry assay exhibited the apoptosis rate of transfected 7721 cells with the lentivirus RNAi vector of Foxp1 or Foxq1 was increased. All the results showed that the lentivirus RNAi vectors of Foxp1 and Foxq1 were able to inhibit the expression of Foxp1 and Foxq1 in 7721 cells efficiently, and the down-regulation of either Foxp1 or Foxq1 resulted in suppression of migration, infiltration and viability of 7721 cells and an increase in cell apoptosis. Our data indicated that both Foxp1 and Foxq1 genes played an oncogenic role in hepatocarcinoma cells, which proposed the two genes as new therapeutic targets for the cancer. [Copyright &y& Elsevier]

Due to the fast mutating nature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of novel therapeutics, vaccines, and evaluating the efficacies of existing one's against the mutated strains is critical for containing the virus. Pseudotyped SARS-CoV-2 viruses are proven to be instrumental in evaluating the efficiencies of therapeutics, owing to their ease in application and safety when compared to handling the live virus. However, a comprehensive protocol that includes selecting transfection reagents, validating different packaging systems for high-throughput screening of neutralizing antibodies, is still a requisite. To this end, we designed and synthesized amide linker-based cationic lipids with varying hydrophilic head groups from dimethyl (Lipo-DME) to methyl, ethylhydroxyl (Lipo-MeOH), and diethylhydroxyl (Lipo-DOH) keeping the hydrophobic tail, stearic acid, as constant. Among the liposomal formulations of these lipids, Lipo-DOH was found to be superior in delivering plasmids and demonstrated comparable transfection efficiencies with commercial standard Lipofectamine 3000. We further used Lipo-DOH for lentivirus and SARS-CoV-2 pseudovirion preparation. For comparing different lentivirus packaging systems, we optimized conditions using Addgene and BEI systems and found that the BEI lenti plasmid system was found to be efficient in making lentiviruses using Lipo-DOH. Using the optimized transfection reagent and the lentivirus system, we developed a robust protocol for the generation of SARS-CoV-2 pseudovirions and characterized their infectivity in human ACE2 expressing HEK-293T cells and neutralizing properties in IgG against spike protein of SARS-CoV-2 positive human sera from individuals recovered from COVID-19. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Deep vein thrombosis (DVT) is one of the common complications of orthopedic surgery. Low molecular weight heparin (LMWH) is a usually used agent for DVT, but it would increase the risk of bleeding. LRRFIP1 has been shown to play an important role in the formation of thrombosis. Therefore, we investigated the effect of LRRFIP1 shRNA lentivirus on DVT in mice. Materials and Methods: Lentiviral Vectors carrying LRRFIP1 shRNA were constructed and transfected into cultured mouse bone marrow cells (BMCs). Male ICR mice were irradiated with a single dose of 9.5Gy and then were injected with different agents through the tail vein. Stasis venous thrombosis was induced by inferior vena cava (IVC) ligation. Mice were sacrificed on the 1st, 3rd and 7th day post operation and the thrombi were removed, blotted the excess blood on it with filter paper and immediately weighed. P-selectin and d-Dimer were determined by enzyme-linked immunosorbent assay (ELISA). Results: LRRFIP1 shRNA significantly suppressed the expression of LRRFIP1 in the thrombi. In contrast, low molecular weight heparin (LMWH) and negative shRNA exhibited little effect on the expression of LRRFIP1. LRRFIP1 shRNA, LMWH and negative shRNA inhibited the thrombus formation in vivo significantly. The plasma P-selectin and d-Dimer levels were significantly increased after IVC ligation. LRRFIP1 shRNA significantly decreased the plasma P-selectin and d-Dimer levels. However, LMWH and negative shRNA showed little effects on the levels of plasma P-selectin and d-Dimer. Conclusion: LRRFIP1 shRNA might represent a promising prevention strategy for DVT. [Copyright &y& Elsevier]

Visna/Maedi virus (VMV), a small ruminant lentivirus responsible for lymphoproliferative pneumonia, encephalitis, arthritis and/or mastitis in sheep, has been detected in different non-lymphoid organs. However, only a few investigations have been carried out in lymphoid tissues. In this study, some lymphoid tissues and lymph node draining or non-draining VMV target organs from five sheep infected experimentally by the respiratory route three years previously were investigated. Archival samples of spleen, red bone marrow, caudal mediastinal lymph nodes, mammary lymph nodes, popliteal lymph nodes and mesenteric lymph nodes were tested by PCR for the presence of proviral DNA. Popliteal and mesenteric lymph node samples were tested also by immunohistochemical staining of the viral capsid antigen p28. The proviral DNA was detected by PCR in all the lymphoid tissue samples from the infected sheep. The viral antigen was stained in mononuclear cells in popliteal and mesenteric lymph nodes of the infected sheep. Although the lymph nodes draining the classical target organs seem to be more infected than the others, both the viral capsid antigen and the proviral DNA were present also in lymph nodes draining non-target organs, such as the mesenteric lymph nodes. These findings show the presence of VMV in different lymphoid tissues in the late stages of infection and suggest a potential role of these tissues as a site for viral reservoir and replication, even three years after infection. [ABSTRACT FROM AUTHOR]

Abstract: The glucocorticoid receptor (GR) and corticotropin-releasing hormone (CRH) are important molecular regulators of an individual’s ability to respond to stressful stimuli in an adaptive manner. Impaired signaling of both GR and CRH often leads to dysfunction of the hypothalamic–pituitary–adrenal axis, which underlies the etiology of many affective disorders such as anxiety and depression. Studies focusing on how GR and CRH influence the stress response are limited as they generalize to broad brain regions, thus hindering identification of how specific CNS nuclei contribute to maladaptive stress responses. Our objective is to distinguish the site-specific involvement of GR and CRH in limbic regions involved in the stress response. With that intent, we use lentiviral (LV) vectors in combination with transgenic mouse lines, enabling us to modify expression of GR or CRH in a very localized manner. This paper describes the generation of several distinct LV vectors and transgenic mice models that will help further elucidate the site-specific actions of GR and CRH. [Copyright &y& Elsevier]

This aim of this paper is to evaluate the presence of Coronavirus in cat faeces and blood samples in order to establish the carriers and eliminators of this virus. The great importance of this carrier state consists in the fact that the cats, specially the ones from the holdhouses and from the owners that have at list two cats in the same house, because the coronavirus is highly contagious, but also can suffer mutations and transformation in feline infectious peritonitis virus which is lethal. We analysed samples from 30 healthy and with clinical signs cats and we observed that the virus was identified only in 4 faces samples using molecular methods like viral ARN extraction and amplification using RT- PCR method. [ABSTRACT FROM AUTHOR]

Abstract: Jembrana disease virus (JDV) is a lentivirus highly related to the bovine immunodeficiency virus (BIV). It causes an acute disease with high mortality rate within 1–2 weeks. JDV encodes the most potent Tat (JTat) of any of the lentiviruses. JTat can transactivate all LTRs and functionally substitute for HIV Tat in the viral genome and may function as a pivotal regulator in the acute pathogenesis of JDV. The goal of this paper is to study JTat internalization by cells, the mechanisms involved in internalization, and the effect of JTat on neighbouring cells. By quantification and fluorescence microscopy, we found that the internalization of extracellular EGFP-JTat fusion protein was both time and dose-dependent, but endocytosis and energy independent. We identified that arginines which were responsible for the internalization. Internalized JTat was distributed in both the nucleus and the cytoplasm, could transactivate JDV LTR and modulate cellular gene expression. Based on our findings, we propose that secretion and internalization of JTat may be a way for JDV to influence neighbouring cells and make the cellular environment more amenable to viral infection. [Copyright &y& Elsevier]

Abstract: α1-Acid glycoprotein (AGP) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this paper we present the feline AGPs (fAGP) glycan moiety modifications in the course of two prevalent feline diseases, the FIV (feline immunodeficiency virus) dependent feline acquired viral immunodeficiency and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect l-fucose residues and Concanavalin A was used to evaluate the degree of branching. It was found that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in FeLV-positive cats diagnosed with lymphoma, while FeLV-positive that did not presented any specific clinical signs cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of fAGP, which however varied widely among individuals. In order to determine the number and the position of oligosaccharide chains, the cDNA sequence of fAGP was also determined. The translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues, with five potential N-glycosylation sites, but also with seven potential phosphorylation sites. [Copyright &y& Elsevier]

The HIV-1 capsid, a conical shell encasing viral nucleoprotein complexes, is involved in multiple post-entry processes during viral replication. Many host factors can directly bind to the HIV-1 capsid protein (CA) and either promote or prevent HIV-1 infection. The viral capsid is currently being explored as a novel target for therapeutic interventions. In the past few decades, significant progress has been made in our understanding of the capsid–host interactions and mechanisms of action of capsid-targeting antivirals. At the same time, a large number of different viral capsids, which derive from many HIV-1 mutants, naturally occurring variants, or diverse lentiviruses, have been characterized for their interactions with capsid-binding molecules in great detail utilizing various experimental techniques. This review provides an overview of how sequence variation in CA influences phenotypic properties of HIV-1. We will focus on sequence differences that alter capsid–host interactions and give a brief account of drug resistant mutations in CA and their mutational effects on viral phenotypes. Increased knowledge of the sequence-function relationship of CA helps us deepen our understanding of the adaptive potential of the viral capsid. [ABSTRACT FROM AUTHOR]

dUTP pyrophosphatase (dUTPase) cleaves the α-β phosphodiester of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for thymine synthesis. Seven crystal structures of feline immunodeficiency virus (FIV) dUTPase in three crystal forms have been determined, including complexes with substrate (dUTP), product (dUMP) or inhibitor (dUDP) bound. The native enzyme has been refined at 1.40 Å resolution in a hexagonal crystal form and at 2.3 Å resolution in an orthorhombic crystal form. In the dUDP complex in a cubic crystal form refined at 2.5 Å resolution, the C-terminal conserved P-loop motif is fully ordered. The analysis defines the roles of five sequence motifs in interaction with uracil, deoxyribose and the α-, β- and γ-phosphates. The enzyme utilizes adaptive recognition to bind the α- and β-phosphates. In particular, the α-β phosphodiester adopts an unfavorable eclipsed conformation in the presence of the P-loop. This conformation may be relevant to the mechanism of α-β phosphodiester bond cleavage. [ABSTRACT FROM AUTHOR]

Presents several abstracts of research papers on medical primatology submitted at the16th Annual Symposium on Nonhuman Primate Models for AIDS held in Atlanta, Georgia on October 7-10, 2004. "Natural infection of chimpanzees with new primate lentiviruses related to HIV-1/SIVCPZ," by J. Takehisa; "Natural infection of a homozygous deleted CCR5 red-capped mangabey with CCRB2-tropic SIV," Z. Chen and colleagues; "Two distinct groups of KSHV-related gamma herpes viruses in macaques," by K. Strand and colleagues.

SERINC5 is a potent lentiviral restriction factor that gets incorporated into nascent virions and inhibits viral fusion and infectivity. The envelope glycoprotein (Env) is a key determinant for SERINC restriction, but many aspects of this relationship remain incompletely understood, and the mechanism of SERINC5 restriction remains unresolved. Here, we have used mutants of HIV-1 and HIV-2 to show that truncation of the Env cytoplasmic tail (ΔCT) confers complete resistance of both viruses to SERINC5 and SERINC3 restriction. Critically, fusion of HIV-1 ΔCT virus was not inhibited by SERINC5 incorporation into virions, providing a mechanism to explain how EnvCT truncation allows escape from restriction. Neutralization and inhibitor assays showed ΔCT viruses have an altered Env conformation and fusion kinetics, suggesting that EnvCT truncation dysregulates the processivity of entry, in turn allowing Env to escape targeting by SERINC5. Furthermore, HIV-1 and HIV-2 ΔCT viruses were also resistant to IFITMs, another entry-targeting family of restriction factors. Notably, while the EnvCT is essential for Env incorporation into HIV-1 virions and spreading infection in T cells, HIV-2 does not require the EnvCT. Here, we reveal a mechanism by which human lentiviruses can evade two potent Env-targeting restriction factors but show key differences in the capacity of HIV-1 and HIV-2 to exploit this. Taken together, this study provides insights into the interplay between HIV and entry-targeting restriction factors, revealing viral plasticity toward mechanisms of escape and a key role for the long lentiviral EnvCT in regulating these processes. [ABSTRACT FROM AUTHOR]

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome. [ABSTRACT FROM AUTHOR]

Background: Small ruminant lentivirus (SRLV) belong to genus Lentivirus, family Retroviridae. These viruses cause caprine arthritis encephalitis (CAE) and maedi visna (MV), infectious diseases that cause economic, production, and reproductive losses. There are no effective treatments or vaccines for these diseases. Thus, early detection via serology has great importance for control of SRLV. Therefore, the objective of this review is to demonstrate the potential of the western blot (WB) test as an immunodiagnostic test for SRLV. Review: In general, immunodiagnosis of SRLV is performed via agar gel immunodiffusion (AGID) and indirect enzymelinked immunosorbent assay (ELISA), which can detect antibodies in several different biological samples but is used preferably with serum and blood plasma. However, WB has demonstrated efficacy in the early diagnosis of immunoglobulins against SRLV, presenting higher sensitivity and specificity than the serological tests usually used, because this technique can detect antibodies at a dilution as much as 256 times greater than that of AGID and 32 times greater than that of ELISA. SRLV infection and consequent immunological activation result in the induction of cellular and humoral responses. Additionally, around the third week, production of antibodies directed mainly toward viral capsid proteins (p25 and p28) occurs. After the fifth week, production of immunoglobulins directed toward other viral proteins occurs. Because of the persistence of SRLV infection, serology is considered to be the most practical means to diagnosis. Each serological test has a percentage specificity and distinct sensitivity, as well as advantages and disadvantages in its applicability. It should be noted that there is no gold standard test for diagnosis of SRLV infection. Moreover, SRLV are characterized by escape mechanisms such as genetic diversity, mutagenic potential, viral intermittence, and the process of compartmentalization, which make immunodiagnosis more difficult. In addition, positive animals tend to present unstable levels of antibodies over weeks, months, and even years. In this context, WB, with early antibody detection, has been proven to be a refined and more accurate technique than other immunodiagnostic tests for SRLV. WB allows the simultaneous resolution of several immunogenic antigens present in a sample, and this feature provides it with greater reliability, differentiates it from other immunological methods, and accredits it as a test of wide applicability. Epidemiological and immunological dynamics studies often use WB in the immunodynamic diagnosis of SRLV. Serum, blood plasma, and seminal plasma are typical biological materials used in the serological diagnosis of SRLV with WB, expanding its potential as an immunodiagnosis method. Conclusion: WB is the most accurate serological technique for SRLV. It is more capable of accurate diagnosis because the genetic diversity that characterizes such lentiviruses and their various immune system escape mechanisms routinely hinder traditional diagnosis. Additionally, this test has been used widely in studies of SRLV for various purposes, but mainly in studies of epidemiological and immunological dynamics, using serum, blood plasma, or seminal plasma. However, independently of the biological sample tested, WB maintains high sensitivity and precision in immunodiagnosis, making it a refined and valid technique for SRLV control programs. [ABSTRACT FROM AUTHOR]

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations. [ABSTRACT FROM AUTHOR]

Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient. [ABSTRACT FROM AUTHOR]