CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers.

Tagging and Capturing of Lentiviral Vectors Using Short RNAs.

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector's envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera.

targeting of lentiviral vectors pseudotyped with the Tupaia paramyxovirus H glycoprotein bearing a cell-specific ligand.

Despite their exceptional capacity for transgene delivery , lentiviral (LV) vectors have been slow to demonstrate clinical utility in the context of applications. Unresolved safety concerns related to broad LV vector tropism have limited LV vectors to applications. Here, we report on a novel LV vector-pseudotyping strategy involving envelope glycoproteins of Tupaia paramyxovirus (TPMV) engineered to specifically target human cell-surface receptors.

A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis.

High-grade urothelial cell carcinoma of the bladder has a poor prognosis when lymph nodes are involved. Despite curative therapy for clinically-localized disease, over half of the muscle-invasive urothelial cell carcinoma patients will develop metastases and die within 5 years. There are currently no described xenograft models that consistently mimic urothelial cell carcinoma metastasis.

Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 components in HEK 293 cells. The Rep 78 and ZFN-based approaches were also compared in HEK 293 cells and in human induced pluripotent stem cells using integrase deficient lentiviral vectors.

Analysis of partial recombinants in lentiviral vector preparations.

The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance.

Acquired substrate preference for GAB1 protein bestows transforming activity to ERBB2 kinase lung cancer mutants.

Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2(YVMA) and ERBB2(G776VC), have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis.

Specific targeting of human interleukin (IL)-13 receptor α2-positive cells with lentiviral vectors displaying IL-13.

The ability to selectively and efficiently target transgene delivery to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. Lentiviral vectors have several advantages that make them attractive as gene delivery vehicles and their tropism can be altered through pseudotyping, allowing transgene delivery to specific populations of cells. The human interleukin-13 receptor α2 (IL-13Rα2) is uniquely overexpressed in many different human tumors, making it an attractive target for cancer therapy.

Production of lentiviral vectors in protein-free media.

The use of lentiviral vectors for transgene delivery in vitro and in vivo for applications in neuroscience, hematology, developmental biology, stem cell biology, and transgenesis has become commonplace. Lentiviral vectors provide an attractive tool for transgene delivery in part because of their ability to incorporate (integrate) into the genomic DNA of target cells with high efficiency, especially in cells that are not actively dividing. In addition, lentiviral vector-mediated transgene )expression can be maintained for long periods of time.

Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus.

Background: The ability to efficiently and selectively target gene delivery vectors to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. We pursued two different strategies to target lentiviral vector delivery to specific cell types. In one of the strategies, vector particles bearing a membrane-bound stem cell factor sequence plus a separate fusion protein based either on Sindbis virus strain TR339 glycoproteins or the vesicular stomatitis virus G glycoprotein were used to selectively transduce cells expressing the corresponding stem cell factor receptor (c-kit).

The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus.

A recombinant live attenuated influenza virus DeltaH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the DeltaH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the DeltaH5N1 vaccine in mice.

Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.

Background: During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.

Methods: Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels.